Heterocyclic-dithiol click chemistry

ABSTRACT

Disclosed are polymers, methods of making polymers, and compositions, focused on cross-linking heterocycles comprising a moiety of Formula I with thiols and thiolates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/530,934, filed Jul. 11, 2017; and U.S. ProvisionalApplication No. 62/680,318, filed Jun. 4, 2018.

FIELD

The present disclosure relates to methods for forming covalent bondsbetween molecules or molecular species of interest.

BACKGROUND

The covalent coupling of two molecules or molecular species is afundamentally important chemical reaction that is widely used in areasas diverse as production and tagging of biomolecules, polymer synthesis,synthesis of complex molecular architectures, such as dendrimers, andsurface chemistry. “Click” chemistry has played an important part inrecent developments in this field. The concept of click chemistry wasfirst introduced in 2001.¹

The term “click” reaction is generally used to describe a chemicalreaction that occurs in one pot, is not sensitive to water, generatesminimal and inoffensive by-products, and has a high thermodynamicdriving force that drives it quickly and irreversibly to a high yield ofa single reaction product, with high reaction specificity. Typical clickreactions include cycloadditions of unsaturated species, such asaddition of azides to alkynes; nucleophilic substitutions; carbonylchemistry of the “non-aldol” type; and additions of carbon-carbonmultiple bonds, including Diels-Alder chemistry.

Click reactions are particularly suitable for carrying out reactions incomplex biological environments in which the reactions need to beregiospecific, high yielding, any by-products need to be non-toxic (forin vivo systems) and the products of the reaction need to bephysiologically stable.

The thiolate group has unique nucleophilicity and polarizatbility,enabling it to be targeted with high specificity. The thiolate-enereaction has a large thermodynamic driving force, has high reactionyields, is stereospecific, and is accepted as a type of clickchemistry.² Numerous drugs form covalent bonds to cysteine (Cys)thiolates. Using submicromolar-affinity kinase inhibitors to minimizeoff-target reactions, -ene, -yne, and other weakly electrophilicwarheads have been used to increase specificity for kinases with aparticular (Cys-containing) structure.³

Thiol-ene reactions are also prevalent in applications requiring thiolcross-linking.⁴ Synthetic applications of thiol-ene cross-linkingreactions include: self-healing polymers,² nanogels,³ thermosettingpolymers, hydrogels,⁵ and dendrimers.⁶ Prevalent biochemicalapplications of thiol-ene cross-linking include functionalizing orstabilizing biotherapeutics in vitro,⁷ and probing high-order proteinstructure and protein-protein interactions.⁸

Dithiolate-diene reactions can also template higher order structures inmaterials ranging from plastics to proteins. Unfortunately, -enemoieties are not bioorthogonal and indiscriminately bind many of thehundreds of cysteines residues in the biological milieu essential forprotein function, for example the lone catalytic cysteine residue inphosphatases and cysteine proteases. This results in levels of toxicitythat limit the in vivo applications of thiol-ene chemistry.

One shortcoming of these thiol-ene cross-linking tools is that they arenot cross-linking selective. These tools will form terminal “dead-end”modifications unless two functional groups happen to be within theirreach.⁹ Dead-end modifications are toxic in vivo; in particular themodification of essential catalytic cysteines (e.g., phosphatases andcysteine [Cys] proteases) and the creation of “non-self” epitopes thatincrease the risk of an adverse immune response.⁹ This inherent toxicityand poor cell permeability have stymied the use of in vivocross-linking.

Thus, there is a need for a biocompatible replacement for dienes indithiolate-diene reactions. Alternatively, or in addition, there is aneed for a chemical tool with improved selectivity for cross-linkingthiols. Alternatively, or in addition, there is a need for new simple,clean, and highly efficient immobilization chemistries, which areapplicable to a broad class of biomolecules.

SUMMARY

In a first aspect, the present disclosure provides a method for covalentcoupling of molecules, the method comprising

providing a plurality of first molecules comprising a moiety of FormulaI:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring, and

contacting the plurality of first molecules with a plurality of secondmolecules comprising at least one thiol functional group,

thereby forming a plurality of covalent bonds between the S or Se atomsof the first molecules and the free thiol groups of the secondmolecules.

In a second aspect, the present disclosure provides a polymer derivedfrom a first monomer and a first cross-linker, wherein

the first monomer comprises at least two thiol functional groups;

the first cross-linker comprises a moiety of Formula I:

-   -   wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring.

In a third aspect, the present disclosure provides a method forproducing a dendrimer, the method comprising:

(i) providing a plurality of first core dendrimer precursor moleculescomprising at least three thiol functional groups; and

contacting the plurality of first core dendrimer precursor moleculeswith a plurality of first cross-linkers comprising a moiety of FormulaI:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; or

(ii) providing a plurality of second core dendrimer precursor moleculescomprising at least three moieties of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; and

contacting the plurality of second core dendrimer precursor moleculeswith a plurality of second cross-linkers comprising at least two thiolfunctional groups,

under conditions to enable a reaction to occur between the thiolfunctional groups and the moieties of Formula I, thereby forming thedendrimer by covalently cross-linking the core precursor dendrimermolecules with the cross-linkers.

In some embodiments, the methods of the first and third aspects, and thepolymers of the second aspect, involve click chemistry reactions becausethey proceed in high yield, with stereoselectivity, at a high reactionrate, and are driven by a thermodynamic driving force.

Advantageously, the moieties of Formula I are more biocompatible thandienes (or other commonly used electrophiles) that have been used tocross-link thiols.

In some embodiments of the first to third aspects,

X is —(CR¹R²)_(n)—, wherein n is an integer selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7 and 8; and

each R¹ and each R² on each CR¹R² group is independently selected fromthe group consisting of: H, OH, NO₂, CN, NH₂, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionallysubstituted C₂-C₁₂alkynyl, optionally substituted C₂-C₁₂heteroalkyl,optionally substituted C₃-C₁₂cycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl,optionally substituted C₆-C₁₈ aryl, optionally substitutedC₁-C₁₈heteroaryl, optionally substituted C₁-C₁₂alkyloxy, optionallysubstituted C₂-C₁₂alkenyloxy, optionally substituted C₂-C₁₂alkynyloxy,optionally substituted C₂-C₁₂heteroalkyloxy, optionally substitutedC₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy,optionally substituted C₁-C₁₂heterocycloalkyloxy, optionally substitutedC₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-C₁₈aryloxy,optionally substituted C₁-C₁₈heteroaryloxy, optionally substitutedC₁-C₁₂alkylamino, CON R³R⁴, NR³COR⁴, NR³COOR⁴, NR³SO₂R⁴, NR³CONR³R⁴, andNR³R⁴, and wherein each R³ and R⁴ is independently selected from thegroup consisting of H, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl,optionally substituted C₂-C₁₂heteroalkyl, optionally substitutedC₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionallysubstituted C₂-C₁₂heterocycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, andoptionally substituted C₁-C₁₈heteroaryl.

In some embodiments of the third aspect, the plurality of first coredendrimer precursor molecules comprising at least three thiol functionalgroups can be formed by a thiol-ene reaction between a dithiol, such as1,3 propane dithiol or 1,4 butane dithiol, with a trialkene, such as2,4,6-tris(allyloxy)-1,3,5-triazine. In some embodiments, this methodcan be used to create “self-healing” dendritic superstructures whoseproperties can be controlled by the ratio of core dendrimer precursormolecules and extender molecules. Alternatively, trithiol compounds andtetrathiol compounds can be cross-linked to provide the core dendrimerprecursor molecule having at least three thiol functional groups. Thecross-linker may comprise moieties of Formula I.

In a fourth aspect, the present disclosure provides a method forproducing a polymer or dendrimer, the method comprising:

providing a plurality of first monomers comprising at least two thiolfunctional groups;

contacting the plurality of first monomers with a plurality of firstcross-linkers comprising a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring,

under conditions to enable a reaction to occur between the thiolfunctional groups and the cross-linker, thereby forming the polymer orthe dendrimer by covalently cross-linking the monomers via thecross-linker molecule.

In a fifth aspect, the present disclosure provides a method of coating asurface of an object, a device, or an assembly, comprising the steps of:

(a) providing a surface of an object, a device, or an assembly;

(b) contacting the surface with a plurality of first moleculescomprising a reactive moiety and a moiety of Formula I

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; and

thereby forming a plurality of covalent bonds between the surface andthe reactive moieties of the first molecule to form a first monolayer.

In an example of the fifth aspect, beta-lipoic acid can be bound to thesurface using the carboxylic moiety. This will form an electrochemicallyactive monolayer (i.e., a first monolayer) that (before electrons areapplied) can perform a click reaction with functionalized dithiols,forming a bilayer (i.e., a first bilayer). In some embodiments, thefirst bilayer is a semiconductor. After electrons are applied thedisulfide bonds can be reduced to create a monolayer (i.e., a secondmonolayer). In some embodiments, the second monolayer will chelatemetals forming a bilayer (i.e., a second bilayer). In some embodiments,the second bilayer is a semiconductor with a P-N gap that is tunable bythe identity of the metal chelated. To the metal layer a third layer ofheterocyclics comprising a moiety of Formula I (e.g., cyclic disulfidesor cyclic thiosulfinates) for a trilayer. In some embodiments, theformation of the trilayer depends upon the redox state of the metals,which can be controlled by an applied voltage. In some embodiments, thetrilayer system is a battery. In some embodiments, the cyclicthiosulfinate acts analogously to the S8 used in second generationlithium ion batteries.

The methods of the first, third and fourth aspects provide reactionsthat and the second aspect provides polymers that can be used to createself-healing polymers, hydrogels, nanogels, thin films, metalnanoparticles, and two layers within a material. In some embodiments,the methods and polymers can be used for functionalizing, preventingcorrosion of, pacifying, and passivating surfaces. In some embodiments,the methods and polymers can be used for synthetic click chemistry. Insome embodiments, the methods and polymers can be used to makephotochromic agents and flavorants. In some embodiments, the methods andpolymers can be used for functionalization of ionic liquids. In someembodiments, the methods and polymers can be used for creatingself-assembled monolayers and molecular scale electronics. In someembodiments, the methods and polymers can be used for superconductors.In some embodiments, the methods and polymers can be used for batteries.

Important features of the reactions occurring in the first, third andfourth aspects, and of polymers of the second aspect, include that: 1)binding to single thiolates is reversible but dithiol cross-linking isnot; 2) the leaving group is only expended upon cross-linking; 3) once adisulfide bond is formed to first thiol moiety, the EC of the secondthiol moiety increases exponentially (i.e., release from the first thiolvia ring closure and forming a cross-link have similar entropy); 4)cross-linking is driven by the considerable bond enthalpies of S—S, S—Seor Se—Se bond and water formation; high yields; and well-establishedstereoselectivity; 5) other reactive functional groups, includingcarboxylates, amines, and disulfides, are avoided.

Reversible cyclic disulfide binding to the transferrin receptor Cys haspreviously been utilized for transport across cell membranes, such asfor drug cargo delivery. On this basis, the compounds of Formula I maybe used for similar applications. Thus, in a sixth aspect, providedherein is a method for transporting a molecule of interest across a cellmembrane comprising:

(a) providing a functionalized molecule of interest comprising a moietyof Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a 3-10-membered cyclic ring; and

(b) contacting a cell of interest with the functionalized molecule ofinterest under conditions for the moiety of Formula I to reversibly bindto one or more cell membrane transport proteins,

thereby facilitating transport of the molecule of interest across thecell membrane.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the present invention will be discussed with reference tothe accompanying figures wherein:

FIG. 1A. Without being bound by any theory, proposed mechanism of thiolcross-linking using cyclic disulfides and cyclic thiosulfinates.Formation of the first disulfide bond is reversible. Dead-endmodification is minimized by entropically favorable ring closure(Mechanism I). Cross-linking proceeds through condensation of CysA, anda sulfenic acid derived from either rate limiting S-oxidation ofthiolateB (Mechanism II) or a cyclic thiosulfinate (Mechanism III).

FIG. 1B. A potential energy surface for the non-enzymatic reactionaccording to the mechanism proposed in FIG. 1A. The free energy valuesare computed using M06-2X/6-311+G(d,p) IEF-PCM^(H2O)//M06-2X/6-31+G(d,p)IEF-PCM^(H2O). *The dotted upper line indicates an activation barrierderived from the zeroth-order half-life kinetics of the oxidation of 5

ΔG=−132.4 kcal mol⁻¹ computed through DFT; ΔG=−131 kcal mol⁻¹ frompublished enthalpies of S—S bond and water formation. Transitionstructures for the nucleophilic addition of MeS⁻ to 1 and 5 are shown.The bond lengths and energy values are reported in Å and kcal mol⁻¹,respectively.

FIG. 2A. Thiol cross-linking by cyclic thiosulfinates kineticallystabilizes the SOD1 dimer in vitro and in cells. Representative raw(left) and deconvoluted (right) mass spectra. The 31,808 Da molecularmass of the cross-linked dimer (D) supports the mechanism proposed inFIG. 1A (e.g., two SOD1 monomers [2×15,844 Da (M)]+1,2-dithiane-1-oxide[136 Da]—oxygen [16 Da]).

FIG. 2B. Representative data of FIG. 2A was used for calculatingcross-linking rates. After 10 min of incubation 1,2-dithiane-1-oxide and1,2-dithiane cross-linked 95% and 0% of SOD1, respectively.

FIG. 2C. Consistent with the rate of cyclic disulfide cross-linkersbeing limited by thiol oxidation 1,2-dithiane cross-links only 11% ofSOD1 after three days Western blot of SOD1 from HepG2 cells incubatedwith various concentrations of compounds for 30 min. EC₅₀s for1,2-dithiane-1-oxide cross-linking were 1-5 μM in HepG2 and HELA cells.

FIG. 3. Determination of 1,2-dithiane-1-oxide cross-linking half-life. Aseries of multi-concentration kinetics experiments was performedfollowing the protocol of Singh et al.¹³ and determined the overallsecond order rate constant of cyclic thiosulfinate-mediatedcross-linking product formation to be 1.5×10⁴ M⁻¹ min⁻¹ which, under theexperimental conditions, extrapolates to a predicted half-life of 3 minfor cross-linking SOD1 with 1,2-dithiane-1-oxide. Shown here arerepresentative results from one concentration, 50 μM SOD1 with 20×excess 1,2-dithiane-1-oxide (1 mM).

FIG. 4. Confirmation of 1,2-dithiane-1-oxide cross-linking site. LC-MSanalysis of WT and CysinSer SOD1 incubated with 1,2-dithiane-1-oxide.SOD1 variant Cys₁₁₁Ser shows no covalent dimer formation confirming thelocation of 1,2-dithiane-1-oxide cross-link at the Cys₁₁₁ pair at SOD1'sdimer interface.

FIG. 5. Complete kinetics of 1,2-dithiane+SOD1 reaction. Incubation of50 μM SOD1 with 20× excess 1,2-dithiane shows slow cross-link formation(compared to cyclic thiosulfinates) at the expected dimer mass of 31,808Da, consistent with the mechanism proposed in FIG. 1A.

FIG. 6. 1,2-dithiane-1-oxide cross-links WT SOD1 in HeLa cells. Westernblots of SOD1 extracted from HeLa cells treated with increasingconcentrations of cyclic thiosulfinate 1,2-dithiane-1-oxide show anapproximately 5 μM EC50 for 1,2-dithiane-1-oxide cross-linking of SOD1.

FIG. 7. 1,2-dithiane-1-oxide cell viability assay. SH-SY5Y cellsincubated with various concentrations of 1,2-dithiane-1-oxide show cellviability is equivalent to untreated cells at cross-linkerconcentrations 50-fold higher than measured EC₅₀, and that the LC₅₀ isc.a. 200-fold that of the EC₅₀.

FIG. 8. The cyclic thiosulfinate, β-lipoic acid cross-links SOD1 in HepG2 cells, and does so more efficiently than the cyclic disulfide,α-lipoic acid. Western blots of SOD1 extracted from Hep G2 treated withincreasing concentrations of lipoic acids (α and β) shows the 1-oxideform increases in vivo cross-linking (as measured by formation of theSOD1 dimer, arrow, following denaturing SDS-PAGE).

FIG. 9. Mass spectrometry assay of α-lipoic acid vs. β-lipoic acidcross-linking of SOD1. Both rate and extent of covalent cross-linking ofSOD1 is increased by oxidation of α-lipoic acid to β-lipoic acid.

FIG. 10A. Cross-linking efficiency of 1,2-dithaine-1-oxide. Incubationof SOD1 with 10×1,2-dithiane-1-oxide.

FIG. 10B. Cross-linking efficiency with 1×1,2-dithiane-1-oxide and 100×reduced glutathione shows that even in the presence of excessglutathione, 1,2-dithiane-1-oxide can efficiently cross-link SOD1. Inthe presence of glutathione the rate of cross-linking was decreased, butthe cross-linking yield (100%) was not affected.

FIG. 11. Mass Spectrometric assay of 1,2-dithiane-1-oxide/DTTcompetition. SOD1 samples incubated for 24 h at various concentrationsof DTT confirm 1,2-dithiane-1-oxide's ability to cross-link the thiolpair of SOD1 at 1:1=cross-linker:DTT.

FIG. 12. The energies of the HOMO and LUMOs of MeS⁻, 1,2-dithiane, and1,2-dithiane-1-oxide, respectively. Computed using M06-2X/6-311++G(d,p)IEF-PCM^(H2O)/M06-2X/6-31+G(d,p).

FIG. 13. Cross-linking SOD1 using 1,2-dithiepane-1-oxide.1,2-dithiepane-1-oxide forms rapid and complete cross-link of SOD1following the same proposed mechanism as 1,2-dithiane-1-oxide while1,2-dithiepane does not. The observed covalent dimer (D) appeared at31,824 Da (two SOD1 monomers [2×15,844 Da]+1,2-dithiepane-1-oxide [152Da]—oxygen [16 Da]).

FIG. 14. Cyclic thiosulfinate-mediated polymerization. All mixturescontained polyethylene dithiol. From left to right mixtures contained nofacilitator (−), Trithiocyanuric acid, Trimethylolpropane Tris(3-MP),and Pentaerythritol tetra(3-MP) and either 1,2-dithiane-1-oxocross-linker or no cross-linker (−). Arrows to solid mass indicatepolymer; arrow to mixture indicates putative nanoparticles.MP=3-mercaptopropionate.

FIG. 15. Cross-linking SOD1 using 1,2-dithiocane-1-oxide.1,2-dithiocane-1-oxide forms rapid and complete cross-link of SOD1following the same proposed mechanism as 1,2-dithiane-1-oxide while1,2-dithiocane does not. The observed covalent dimer (D) appeared at31,834 Da (two SOD1 monomers [2×15,844 Da]+1,2-dithiocane-1-oxide [162Da]—oxygen [16 Da]).

DETAILED DESCRIPTION

Disclosed herein are polymers, methods of making polymers, andcompositions, focused on a more biocompatible method for cross-linkingthiols and thiolates. In some embodiments of the present disclosure,polymers, methods of making, and compositions focus on the reaction ofdithiols and dithiolates with heterocycles. In some embodiments of thepresent disclosure, polymers, methods of making, and compositions focuson the reaction of thiols and thiolates with heterocycles. In someembodiments, the heterocycles comprise a moiety of Formula I

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring. In cyclic disulfides, W and Y are S. Insome embodiments, the heterocycle is a cyclic thiosulfinate, wherein Wis S and Y is S(O). In some embodiments, the heterocycle is a cyclicdiselenide, wherein W and Y are Se. In some embodiments, the heterocycleis a cyclic selenoselenoxide, wherein W is Se and Y is Se(O). In someembodiments, the heterocycle is a cyclic selenosulfide, wherein W is Seand Y is S; or W is S and Y is Se. In some embodiments, the heterocycleis a cyclic selenosulfinate, wherein W is Se and Y is S(O). In someembodiments, the heterocycle is a cyclic thioselenoxide, wherein W is Sand Y is Se(O).

Thiols, thiolates, dithiols, and dithiolates are used in a variety ofchemical reactions. In some embodiments, the heterocycle comprising amoiety of Formula I is used to replace an electrophile (e.g., an -enemolecule, an -yne molecule, a Michael acceptor, etc.) inthiol-electrophile mediated cross-linking. In some embodiments, theheterocycle comprising a moiety of Formula I reacts with a thiol, athiolate, a dithiol, or a dithiolate in the cross-linking ofself-healing polymers,⁸ hydrogels,⁹ nanogels,¹⁰ thin films,¹¹ metalnanoparticles,¹² and two layers within a material.¹³ In someembodiments, the reactions and methods disclosed herein are used infunctionalizing,¹⁴ preventing corrosion of,¹⁵ pacifying,¹⁶ andpassivating surfaces. In some embodiments, the reactions and methodsdisclosed herein are used for synthetic click chemistry (e.g., use adithiol such as benzene dithiol to react with oxo-cyclic disulfides). Insome embodiments, the reactions and methods disclosed herein catalyzeclick chemistry (serving as Cu ligand).¹⁷ In some embodiments, thereactions and methods disclosed herein are used to make photochromicagents¹⁸ and flavorants. In some embodiments, the reactions and methodsdisclosed herein are used for the functionalization of ionic liquids.¹⁹In some embodiments, the reactions and methods disclosed herein are usedto create self-assembled monolayers and molecular scale electronics.²⁰In some embodiments, the reactions and methods disclosed herein are usedfor superconductors.²¹ In some embodiments, the reactions and methodsdisclosed herein are used for capacity retention in lithium-sulfurbatteries.²² The most prevalent mechanisms employed for in vitropolymerization and surface chemistry are thiol-ene click chemistry (bothradical- and Michael-types) and metal ligation, respectively.

In some embodiments, the reactions and methods disclosed herein areanalogous to using dienes, wherein the diene is replaced by a morebiocompatible heterocycle comprising a moiety of Formula I (e.g., acyclic disulfide). The methods described herein differ from that ofBarcan²³ whenever a cyclic disulfide is used as the group of Formula I,and when the cyclic disulfide is not used as the cross-linker, itdiffers from Barcan's methods by employing one dithiol per cylicdisulfide, which therefore does not result in the formation of any freethiol that need to be capped/functionalized (in Barcan's case usingmaleimide thiol-ene chemistry).

The reactions and methods described herein address the need for chemicaltools that can selectively form cross-links. Contemporarythiol-selective cross-linkers, for example, modify all accessiblethiols, but only form cross-links between a subset. The resultingterminal “dead-end” modifications of lone thiols are toxic, confoundcross-linking-based studies of macromolecular structure, and are anundesired—and currently unavoidable—by-product in polymer synthesis.

As exemplified by thiolate-ene “click” chemistry, the unique propertiesof thiolates enable highly selective reactions. Dithiol-diene reactionpartners can template high order structures and are employed (in vitro)in polymer syntheses, in biochemical probes, and to stabilize proteins.Unfortunately, enes react with a myriad of essential protein cysteineresidues, resulting in toxicity that limits their applicability in vivo.A more biocompatible and/or selective method for cross-linking thiolatepairs was sought. It was reasoned that cyclic thiosulfinates—S-oxoderivatives of well-tolerated cyclic disulfide natural products—wouldselectively cross-link dithiols. Without being bound by any theory, thefollowing mechanism is proposed: a nucleophilic attack by thiolate A(e.g., CysA) upon the thiosulfinate disulfide results inthiolate-disulfide interchange concomitant to ring cleavage, and adisulfide tethered terminal sulfenic acid moiety (FIG. 1A). Next, anucleophilic attack by thiolate B on the sulfenic sulfur results in theformation of a second disulfide bond coupled to the release of water anda cross-link (FIG. 1A). It was also reasoned that a similar mechanismwould occur with cyclic selenoselenoxides, cyclic thioselenoxides andcyclic selenosulfinates.

In some embodiments, important attributes of dithiol-cyclicthiosulfinate chemistry include: 1) avoiding binding to lone cysteines(for example the proteins in a human that one does not want across-linker to react with) is reversible, whereas cross-linking ofdithiols is not; 2) the cross-linking step is driven by the considerablebond enthalpies of disulfide bond and water formation; 3) most of theattributes of click chemistry including orthogonality with other proteinfunctional groups, high reaction yields, rapid reactions,stereoselectivity, compatibility with aqueous solvents, and a ringstrain-dependence that is orders of magnitude higher than existingperiod 2 element systems.

Using the thiol pair of Cu/Zn-superoxide dismutase (SOD1) it wasdemonstrated that cyclic disulfides—including the drug/nutritionalsupplement lipoic acid—efficiently cross-linked thiol pairs but avoideddead-end modifications. Thiolate-directed nucleophilic attack upon thecyclic disulfide resulted in thiol-disulfide exchange and ring cleavage.The resulting disulfide-tethered terminal thiolate moiety eitherdirected the reverse reaction, releasing the cyclic disulfide, orparticipated in oxidative disulfide (cross-link) formation (FIG. 1A). Itwas hypothesized—and confirmed with density functional theory (DFT)calculations—that mono-S-oxo derivatives of cyclic disulfides formed aterminal sulfenic acid upon ring cleavage that obviated the previouslyrate-limiting step, thiol oxidation, and accelerated the newrate-determining step, ring cleavage. Without being bound by any theory,the DFT calculations suggest that the origin of accelerated ringcleavage is improved frontier molecular orbital overlap in thethiolate-disulfide interchange transition (FIG. 1). Five toseven-membered cyclic thiosulfinates were synthesized and efficientlycross-linked up to 104-fold faster than their cyclic disulfideprecursors; functioned in the presence of biological concentrations ofglutathione; and acted as cell-permeable, potent, tolerable,intracellular cross-linkers. In some embodiments, this new class ofthiol cross-linkers exhibited click-like attributes including, highyields driven by the enthalpies of disulfide and water formation,orthogonality with common functional groups, water-compatibility, andring strain-dependence.

The reactions require that binding to lone thiolates be reversible andideally favorable (FIG. 1A, top). It was reasoned that the higheffective concentration (“EC”) of cyclic disulfides—i.e., theirentropically driven propensity to remain oxidized and cyclic—wouldenable reversible binding to a single thiolate. Covalent binding of acyclic disulfide to a thiolate can proceed rapidly by S_(N) ²thiolate-disulfide interchange. This has been utilized for drug cargodelivery—cargos are transported across the cell membrane via reversiblecyclic disulfide binding to the transferrin receptor Cys.²⁴ Importantly,the ring strain- (in particular the C—S—S—C dihedral angle-) dependenceof cyclic disulfide reactivity (and EC) is much greater than that ofrings composed of period-two elements. ²⁵ In other words, cyclicdisulfide reactivity is predictable and highly tunable.

Thus, also provided herein is a method for transporting a molecule ofinterest across a cell membrane, comprising:

(a) providing a functionalized molecule of interest comprising a moietyof Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; and

-   -   (b) contacting a cell of interest with the functionalized        molecule of interest under conditions for the moiety of Formula        I to reversibly bind to one or more cell membrane transport        proteins,

thereby facilitating transport of the molecule of interest across thecell membrane.

In some embodiments, the cell membrane transport protein is anATP-powered pump protein, an ion channel protein, or a transporterprotein. In some embodiments, the cell membrane transport protein istransferrin receptor.

A cyclic disulfide cannot cross-link dithiolates without an oxidant—anadditional two electron oxidation is be required to form the seconddisulfide bond (FIG. 1A, middle). It was reasoned that if S-oxo-cyclicdisulfides (cyclic thiosulfinates) were used, the oxo group could leaveas water concomitant to formation of the second disulfide andirreversible cross-link (FIG. 1A, bottom). In summary, nucleophilicattack by thiolateA upon the cyclic thiosulfinate results inthiolate-disulfide interchange. A terminal sulfenic acid is exposed,which is then be attacked by thiolateB. The sulfenic oxygen is reducedconcomitant to oxidative formation of the second disulfide, resulting ina dithiolate cross-link.

Thus, provided herein is a method for covalent coupling of molecules.The method comprises providing a plurality of first molecules comprisinga moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; and contacting the plurality of firstmolecules with a plurality of second molecules comprising at least onethiol functional group, thereby forming a plurality of covalent bondsbetween the S or Se atoms of the first molecule and the free thiolgroups of the second molecule.

In some embodiments, W and Y are S in Formula I. In these embodiments,the reaction may be generally represented as:

where R and R′ are molecules of interest or linker groups attached tomolecules of interest.

In some embodiments, W is S and Y is S(O) in Formula I. In theseembodiments, the reaction may be represented as:

wherein R and R′ are molecules of interest or linker groups attached tomolecules of interest.

In each of the reactions above, one or more of the sulfur atoms on thegroup of Formula I can be interchanged with a selenium atom.

The reaction is carried out under conditions to enable a reaction tooccur between the thiol groups and the moieties of Formula I, therebycovalently cross-linking the first molecule to the second molecule. Insome embodiments, a plurality of covalent bonds between the plurality ofthe S or Se atoms of the first molecule and the plurality of free thiolgroups of the second molecule is formed.

When W and Y are S or Se in Formula I (i.e., a cyclic disulfide orcyclic diselenide), the conditions will generally comprise treating thereaction mixture with an oxidizing agent. Peracids and peroxides may beused for this purpose. A range of oxidizing agents that can be used tooxidize sulfur or selenium are known in the art and can be used.Examples include, but are not limited to: m-chloroperoxybenzoic acid(mCPBA), and hydrogen peroxide.

The conditions may comprise contacting the two molecules of interestwith one another in a suitable solvent. Suitable solvents include, butare not limited to, water, DMSO, DMF, methanol, ethanol, propanol,dichloromethane, and mixtures thereof.

In some embodiments, 1,2-dithianes and 1,2-dithiane-1-oxides are used inthe polymers, methods of making polymers, and compositions describedherein. In some embodiments of the moiety of Formula I as describedabove and below,

X is —(CR¹R²)_(n)—, wherein n is an integer selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7 and 8; and

each R¹ and each R² on each CR¹R² group is independently selected fromthe group consisting of: H, OH, NO₂, CN, NH₂, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionallysubstituted C₂-C₁₂alkynyl, optionally substituted C₂-C₁₂heteroalkyl,optionally substituted C₃-C₁₂cycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl,optionally substituted C₆-C₁₈ aryl, optionally substitutedC₁-C₁₈heteroaryl, optionally substituted C₁-C₂alkyloxy, optionallysubstituted C₂-C₁₂alkenyloxy, optionally substituted C₂-C₁₂alkynyloxy,optionally substituted C₂-C₁₂heteroalkyloxy, optionally substitutedC₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy,optionally substituted C₁-C₁₂heterocycloalkyloxy, optionally substitutedC₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-C₁₈aryloxy,optionally substituted C₁-C₁₈heteroaryloxy, optionally substitutedC₁-C₁₂alkylamino, CON R³R⁴, NR³COR⁴, NR³COOR⁴, NR³SO₂R⁴, NR³CONR³R⁴, andNR³R⁴; and wherein each R³ and R⁴ is independently selected from thegroup consisting of: H, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl,optionally substituted C₂-C₁₂heteroalkyl, optionally substitutedC₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionallysubstituted C₂-C₁₂heterocycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, andoptionally substituted C₁-C₁₈heteroaryl.

In some embodiments of the moiety of Formula I as described above andbelow, W and Y are S. In some other embodiments, W is S and Y is S(O).In some other embodiments, W and Y are Se. In some other embodiments, Wis Se and Y is Se(O). In some other embodiments, W is Se and Y is S. Insome other embodiments, W is S and Y is Se. In some other embodiments, Wis Se and Y is S(O). In some other embodiments, W is S and Y is Se(O).

In some embodiments as described above and below, W and Y are S, and themoiety of Formula I is represented as follows:

wherein n is an integer from 1 to 8.

In some embodiments as described above and below, W is S and Y is S(O),and the moiety of Formula I is represented as follows:

wherein n is an integer from 1 to 8.

In some embodiments as described above and below, W is S and Y is S(O)₂,and the moiety of Formula I is represented as follows:

wherein n is an integer from 1 to 8.

In some embodiments of the moiety of Formula I as described above andbelow, X is —(CR¹R²)_(n)— where R¹ and R² are as described earlier and nis an integer selected from the group consisting of 3, 4 or 5. In someembodiments, R¹ and R² are H. In some embodiments, the moiety of FormulaI is selected from the group consisting of the following formulae (wherethe wavy line represent the point of attachment to a molecule ofinterest):

In some embodiments of the moiety of Formula I as described above andbelow, X is —(CR¹R²)_(n)— where R¹ and R² are as described earlier and nis an integer selected from the group consisting of 3, 4, 5, 6, or 7. Insome embodiments, the moiety of Formula I is selected from the groupconsisting of:

In some embodiments, the moiety of Formula I is selected from the groupconsisting of:

In some embodiments of the moiety of Formula I as described above andbelow, each R and each R² on each CR¹R² group is independently selectedfrom the group consisting of H, OH, NO₂, CN, NH₂, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂heteroalkyl, optionallysubstituted C₃-C₁₂cycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkyl, optionally substituted C₁-C₁₂alkyloxy,optionally substituted C₂-C₁₂heteroalkyloxy, optionally substitutedC₃-C₁₂cycloalkyloxy, and optionally substitutedC₁-C₁₂heterocycloalkyloxy.

In some embodiments of the moiety of Formula I as described above andbelow, each R¹ and each R² on each CR¹R² group is independently selectedfrom the group consisting of H, OH, NO₂, CN, NH₂, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₁-C₁₂alkyloxy, and optionally substitutedC₃-C₁₂cycloalkyloxy.

In some embodiments as described above and below, the heterocyclecomprising a moiety of Formula I is selected from the group consistingof:

In some embodiments of the moiety of Formula I as described above andbelow, one or more of the sulfur (S) atoms in the structures above couldbe interchanged with a selenium (Se) atom, and the resultant structuresare also contemplated by the present disclosure.

In some embodiments, the methods and materials described herein are usedin “click” chemical reactions. For example, the pairing of the groupFormula I and the dithiol (or dithiolate) can replace the azide-alkynepair and/or the tetrazine-alkene/alkyne pairs used in known clickchemical reactions. In some embodiments, the molecule used in themethods described herein comprises a moiety selected from the groupconsisting of, but not limited to: dyes (e.g., fluorescent dyes,non-fluorescent dyes, quencher dyes); tags (e.g., biotin, FLAG tag);bifunctional reagents; trifunctional reagents; PEGylation reagents;biomolecules (e.g., nucleotides, nucleosides, amino acids, RNA, DNA,peptides, proteins, monosaccharides, polysaccharides); and substrates(e.g., agarose, magnetic beads).

In some embodiments, the methods and materials described herein areregiospecific. In some embodiments, the methods and materials describedherein are stereospecific. In some embodiments, the methods andmaterials described herein produce or represent a single isomer. In someembodiments, the methods and materials described herein produce orrepresent a mixture of isomers.

The methods and materials described herein can also be used to formdendrimers. In the work of Fang²⁶, a cyclic disulfide was reduced priorto use and then thiol-ene cross-linkers were used. In contrast, in thepresent methods the use of oxo cyclic disulfides in place of the cyclicdisulfide are cross-linked using molecules with (bifunctional) pair ofdithiols. Alternatively, the cyclic disulfide could be reduced to adithiol, and then reacted with (for example) a homobifunctional cyclicthiosulfinate cross-linker (or more slowly with homobifunctional cyclicdisulfide cross-linkers). Alternatively, the cyclic disulfide abovecould be replaced by a monothiol, and two such molecules could becross-linked with a single cyclic disulfide or cyclic thiosulfinate. Insome embodiments, a cyclic disulfide is used in combination with anoxidizing agent.

In addition, the methods and materials described herein can also be usedin cosmetic treatments. Keratin-containing materials consists of manylong protein chains that are bound to each other via 1) hydrogenbonding, 2) salt bridges between acid and base groups, and 3) disulfidebonds. Disulfide bonds in keratin-containing materials are broken at aslightly alkaline pH of 8.5, due to heating or use of various reducingtreatments. For keratin-containing material treatment, the methods andmaterials described herein can be used to treat keratin-containingmaterials by cross-linking the free thiol groups in keratin-containingmaterials. For example, the compound of Formula I may be attached to ahair treatment agent, such as a colorant, a therapeutic agent or acosmetic agent, and hair containing broken disulfide bonds may betreated with a composition containing the hair treatment agent tothereby cross link the thiol groups and/or covalently attend the hairtreatment agent to the hair.

In addition, in vivo thiol cross-linking using the methods describedherein can be used as a strategy for pharmacological proteinstabilization, and a long-sought, non-inhibitory alternative tostabilization with substrate analogues.¹¹ A number of diseases,including familial Amyotrophic Lateral Sclerosis (fALS), are associatedwith loss of quaternary structure and protein destabilization (seen withCu/Zn-superoxide dismutase (SOD1) mutations). Multimerstabilization—exemplified by the substrate/cargo analogue,transthyretin-stabilizing drug tafamidis¹²—is a therapeutic strategy inthese diseases. Thiol-ene cross-linkers were used in a proof-of-conceptstudy to demonstrate that cross-linking the thiol pair (Cys_(111A+B), 8Å apart) on adjacent subunits of SOD1 could stabilize fALS-SOD1 variantsby up to 40° C.⁸ This approach also rescued the enzymatic activity ofinherently inactive fALS SOD1 variants.⁸ To determine if this approachwas applicable to other proteins, a computational screen of humanprotein structures was performed. This screen discovered 20 additionalmultimeric proteins with quaternary structures that could be stabilizedby intersubunit cysteine cross-linking including DJ-1, a dimeric proteindestabilized by Parkinson's associated mutations.

The present inventors surveyed drugs to identify mechanisms forselective thiol binding that can be tolerated in vivo. One recentapproach to drug design is to attach a soft, sometimes finely “tuned”Cys-selective electrophile¹⁰ to a high-affinity binder.¹³⁻¹⁴Unfortunately, as is often the case, the lack of high-affinity SOD1binders ruled out this structure-based approach. The other mechanismused by thiolate-selective drugs, disulfide bond formation, is the mostprevalent and mature (disulfiram/Antabuse treatments began in 1948).¹⁵Inactive prodrugs are transformed into thiols, which, after spontaneousoxidation, form long-lived disulfide bonds between the drug metabolite'ssulfenic acid and a target protein's cysteine thiolate. Some drugs formdisulfides with enzyme active site Cys (e.g., disulfiram¹⁶) and otherswith allosteric Cys (e.g., omeprazole/Prilosec,¹⁷ prasugrel/Effiant,¹⁸etc.). Unfortunately, the strategy of binding two of these drugs tocreate a bifunctional cross-linker would not result in a tool that couldavoid dead-end modifications.

In some embodiments, the heterocycles comprising a moiety of Formula Idisclosed herein form transient bonds. In some embodiments, theheterocycles avoid dead-end modifications without additional molecules.In some embodiments, the heterocycles are cyclic disulfides or cyclicthiosulfinates. Cyclic disulfides are the only thiolate-selectivescaffold that the inventors are aware of that can form transient bonds(i.e., can avoid dead-end modifications without the aid of othermolecules). Cyclic disulfide chemistry was extensively characterized ina series of publications by the Whitesides group.¹⁹⁻²¹ These studiesdemonstrated the high effective concentration (EC—i.e., theentropically-driven propensity to remain oxidized and cyclic;specifically the K_(eq) between a dithiol forming a cyclic disulfide anda dialkyl disulfide forming two thiols) of cyclic disulfides results intransient binding to lone thiols. Moreover, the K_(eq) of cyclicdisulfide binding to lone thiols (i.e., “K_(eq) dead-end”) is highlyring strain-dependent, varying over three orders of magnitude.¹⁹⁻²⁰Cyclic disulfide-tethered drug cargos can even be transported—with anefficiency that varies with ring strain-across the cell membrane viareversible binding to a transferrin receptor Cys.^(22,27) Cyclicdisulfides can be tolerated at doses up to 5 g/day/person and have anLD₅₀ in the range of ethanol, fructose, and sodium chloride.

The present inventors postulated that cyclic disulfides could cross-linkthiol pairs while minimizing dead-end Cys modifications. A reversibleS_(N)2-type attack of a Cys thiolate upon a cyclic disulfide wouldresult in thiolate-disulfide interchange concomitant to ring opening toform a terminal thiolate. If this terminal thiolate was within bindingdistance of a sulfenate (i.e., oxidized Cys), a cross-link could form bytheir condensation to a disulfide bond (FIG. 1A, Mechanism II).²⁸Otherwise, the cyclic disulfide would be released by the reverse(thiolate-disulfide interchange) reaction (FIG. 1A, Mechanism I).Furthermore, if mono-S-oxo cyclic disulfides (cyclic thiosulfinates)were used instead, thiolate oxidation, the slowest step of thecross-linking reaction sequence, would not be required (FIG. 1A,Mechanism III). Instead, thiolate-disulfide interchange with a cyclicthiosulfinate would lead directly to a disulfide-bound terminal sulfenicacid, which would rapidly form a cross-link by condensing with thesecond, nearby thiolate, releasing water.²⁸ Thiolate-disulfideinterchange proceeds through a linear trisulfide-like intermediatecomprised of nucleophilic-(S_(n)), center-(S_(c)), and leavinggroup-(S_(l)) sulfurs with Brønstead coefficients (β) of −0.5, −0.3, and−0.7, respectively.^(20,29) The Brønstead coefficients of −0.7 and −0.3for the leaving group and central sulfur, respectively, and our quantummechanical calculations (FIG. 1B) imply that the rate ofthiolate-disulfide interchange is highly sensitive and inverselyproportional to the pK_(a) of S_(l). DFT calculations were used tounderstand the origins of the nearly 110-fold reactivity increase of 1towards lone thiolates over 5 and the observed reversibility. Aconformational search was performed on the starting materials,transition structures, and intermediates. Glutathione was represented asmethyl thiolate to reduce the conformational search space andcomputation time.

The 2.9 kcal mol⁻¹ lower activation free energy of TS(1→2b) vs. TS(5→2a)is due to more favorable frontier molecular orbital interactions in thetransition state (FIG. 1B). The σ* orbital (FIG. 12) of 1 is 0.21 eVlower in energy than that of 5, thus lowering the energy of TS(1→2b).The ring-opening step for 1 and 5 are endergonic (ΔG=4.6 and 5.4 kcalmol⁻¹, respectively) and reversible, consistent with experiments. Theformation of the cross-linked product and water is thermodynamicallyfavored, over 130 kcal mol⁻¹ lower in free energy than the reactants.Upon ring-opening of 1,2-dithiane, 2a is slowly oxidized (t_(1/2)=10days) to 3a, which is rate-determining and affords the finalcross-linked product. The QM results highlight two major implicationsfor the low pK_(a) sulfenic acid group. First, the nucleophilic attackon the non-oxo-thiosulfinate S, which releases sulfenic acid,is >10-fold faster and therefore more likely than the attack on the moreelectrophilic sulfinyl sulfur. Second, in addition to eliminating theneed for rate-limiting thiol oxidation, thiosulfinates, through thesulfenate intermediate generated, also increase the rate ofthiolate-disulfide interchange.

Following the general protocols described herein, a polymer or adendrimer can also be produced by:

providing a plurality of first monomers comprising at least two thiolfunctional groups;

contacting the plurality of first monomers with a plurality of firstcross-linkers comprising a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring, under conditions to enable a reactionto occur between the thiol functional groups and the cross-linker,thereby forming the polymer or the dendrimer by covalently cross-linkingthe monomers via the cross-linkers.

In another aspect, provided herein is a polymer derived from a firstmonomer and a first cross-linker, wherein

the first monomer comprises at least two thiol functional groups;

the first cross-linker comprises a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring.

In certain embodiments of the preceding Formula I, Y is independentlyselected from the group consisting of S(O), Se(O), S(O)₂ and Se(O).

In some embodiments of the methods and polymers disclosed above andbelow, the first monomer is selected from the group consisting of adithiol compound, a trithiol compound, a tetrathiol compound, and athiomer. In some embodiments, the first monomer is selected from thegroup consisting of a dithiol compound, a trithiol compound, atetrathiol compound, a hexathiol compound, and an octathiol compound.

In some embodiments of the methods and polymers disclosed above andbelow, the first monomer is selected from the group consisting ofdithiothreitol (DTT), 1,2-ethanedithiol, 1,3-propanedithiol,1,4-butanedithiol, 1,5-pentanedithiol, 1,6-hexanedithiol,1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,1,10-decanedithiol, 1,11-undecanedithiol, 1,12-dodecanedithiol,1,13-tridecanedithiol, 1,14-tetradecanedithiol, 1,16-hexadecanedithiol,dithiolbutylamine (DTBA), tetra(ethylene glycol) dithiol, hexa(ethyleneglycol) dithiol, 2-mercaptoethyl ether, 2,2′-thiodiethanethiol,2,2′-(ethylenedioxy)diethanethiol, propane-1,2,3-trithiol,trimethylolpropane tris(2-mercaptoacetate), trimethylolpropanetris(3-mercaptoacetate),3,3′,3″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(propane-1-thiol),4,4′,4″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))-tris(butane-1-thiol),pentaerythrityl tetrathiol, pentaerythritoltetrakis(3-mercaptopropionate), a peptide having a disulfide bond, and aprotein having a disulfide bond.

In some embodiments, the first monomer is a peptide having a disulfidebond or a protein having a disulfide bond. In some embodiments, thefirst monomer is a protein having a disulfide bond.

In some embodiments, the first monomer is copper/zinc superoxidedismutase (SOD1) or DJ-1, which has been implicated in Parkinson'sdisease. In some embodiments, the first monomer is selected from thegroup consisting of an SOD1 variant, a DJ-1 variant, an effector caspase(e.g., Caspase-3), and iron-sulfur cluster assembly enzyme (IscU). Insome embodiments, the first monomer is SOD1. In some embodiments, thefirst monomer is a fALS-SOD1 variant. In some embodiments, the firstmonomer is a DJ-1 variant. In some embodiments, the first monomer is aneffector caspase. In some embodiments, the first monomer is Caspase-3.In some embodiments, the first monomer is IscU.

In some embodiments, the first monomer is a keratin-containing material.In some embodiments, the keratin-containing material is selected fromthe group consisting of hair (including facial hair such as eyebrows,eyelashes, beards, and moustaches), fingernails and toenails. In someembodiments, the keratin-containing material is selected from the groupconsisting of hair, eyebrows, eyelashes, fingernails and toenails. Insome embodiments, the keratin-containing material is hair.

In some embodiments of the methods and polymers disclosed above andbelow, the first monomer is selected from the group consisting ofpoly(ethylene glycol) dithiol (PEG-DT, e.g., with average M_(n) of1,500), 4arm-PEG2K-SH, 4arm-PEG5K-SH, 4arm-PEG10K-SH, 4arm-PEG20K-SH,4-arm poly(ethylene oxide) thiol-terminated, 8arm-PEG10K-SH (hexaglyerolcore), 8arm-PEG10K-SH (tripentaerythritol core), 8arm-PEG20K-SH(hexaglyerol core), 8arm-PEG20K-SH (tripentaerythritol core), and 8-armpoly(ethylene oxide) thiol-terminated. In some embodiments of themethods and polymers disclosed above and below, the first monomer ispoly(ethylene glycol) dithiol or polyethylene dithiol.

In certain embodiments of the methods and polymers disclosed above andbelow, the first monomer comprising at least two thiol functional groupsis a polyalkylene dithiol, such as polyethylene dithiol, polypropylenedithiol, etc.

In some embodiments in the methods and polymers disclosed above andbelow, the first cross-linker comprising a moiety of Formula Istabilizes a first monomer. In some embodiments, the first cross-linkerstabilizes a peptide having a disulfide bond or a protein having adisulfide bond. In some embodiments, the first cross-linker stabilizes afALS-SOD1 variant or a DJ-1 variant. In some embodiments, the unfoldingtemperature of the first monomer is increased. In some embodiments, theunfolding temperature is increased by at least 2° C., at least 5° C., atleast 10° C., at least 15° C., at least 20° C., at least 25° C., atleast 30° C., at least 35° C., at least 40° C., at least 45° C., atleast 50° C., at least 55° C., at least 60° C., at least 65° C., atleast 70° C., at least 75° C., at least 80° C., at least 85° C., atleast 90° C., at least 95° C., or at least 100° C. In some embodiments,the unfolding temperature is increased by an amount selected from thegroup consisting of about 1° C., about 2° C., about 3° C., about 4° C.,about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about10° C., about 11° C., about 12° C., about 13° C., about 14° C., about15° C., about 16° C., about 17° C., about 18° C., about 19° C., about20° C., about 21° C., about 22° C., about 23° C., about 24° C., about25° C., about 26° C., about 27° C., about 28° C., about 29° C., about30° C., about 31° C., about 32° C., about 33° C., about 34° C., about35° C., about 36° C., about 37° C., about 38° C., about 39° C., about40° C., about 41° C., about 42° C., about 43° C., about 44° C., about45° C., about 46° C., about 47° C., about 48° C., about 49° C., about50° C., about 51° C., about 52° C., about 53° C., about 54° C., about55° C., about 56° C., about 57° C., about 58° C., about 59° C., about60° C., about 61° C., about 62° C., about 63° C., about 64° C., about65° C., about 66° C., about 67° C., about 68° C., about 69° C., about70° C., about 71° C., about 72° C., about 73° C., about 74° C., about75° C., about 76° C., about 77° C., about 78° C., about 79° C., about80° C., about 81° C., about 82° C., about 83° C., about 84° C., about85° C., about 86° C., about 87° C., about 88° C., about 89° C., about90° C., about 91° C., about 92° C., about 93° C., about 94° C., about95° C., about 96° C., about 97° C., about 98° C., about 99° C., about100° C., about 101° C., about 102° C., about 103° C., about 104° C.,about 105° C., about 106° C., about 107° C., about 108° C., about 109°C., and about 110° C.

In some embodiments in the methods and polymers disclosed above andbelow, the first cross-linker comprising a moiety of Formula Isuppresses aggregation of a first monomer. In some embodiments, thefirst cross-linker suppresses aggregation of a peptide having adisulfide bond or a protein having a disulfide bond. In someembodiments, the first cross-linker suppresses aggregation of afALS-SOD1 variant.

In some embodiments in the methods and polymers disclosed above andbelow, the first cross-linker comprising a moiety of Formula I inhibitsapoptosis. In some embodiments, the first cross-linker cross-links aneffector caspase.

In some embodiments in the methods and polymers disclosed above andbelow, the first cross-linker comprising a moiety of Formula I causesferroptosis. In some embodiments, the first cross-linker kills aplurality of cancer cells. In some embodiments, the first cross-linkercross-links IscU.

In some embodiments in the methods and polymers disclosed above andbelow, the first cross-linker comprising a moiety of Formula I is usedin a method for treating a keratin-containing material. In someembodiments, the first cross-linker cross-links the keratin-containingmaterial. In some embodiments, the method for treating akeratin-containing material, comprises:

-   -   i) providing a plurality of first monomers, wherein the first        monomer is a keratin-containing material sample comprising a        plurality of disulfide bonds;    -   ii) applying to the keratin-containing material sample for a        period of time a mixture, comprising a reducing agent in a        concentration of about 0.1% by weight to about 15% by weight,        thereby producing a reduced keratin-containing material sample,        wherein the reduced keratin-containing material sample comprises        a plurality of free thiol groups; and    -   iii) applying a first cross-linker to the reduced        keratin-containing material sample, wherein the first        cross-linker comprises a moiety of Formula I

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring,

thereby forming a plurality of covalent bonds between the free thiolgroups and the first cross-linkers.

In some embodiments, the reducing agent is selected from the groupconsisting of ammonium thioglycolate, L-cysteine, glutathione, ascorbicacid, beta-mercaptoethanol, 2-mercaptoethylamine, 2-mercaptoethylaminehydrochloride, dithiothreitol (DTT), thiolactic acid, thiosalicylicacid, tris-2-carboxyethylphospine hydrochloride (TCEP), sodiumhydrosulfite, sodium thiosulfate, potassium disulfite, sodium disulfite,sodium bisulfate, sodium bisulfite, ammonium bisulfite, thioglycolicacid, calcium thioglycolate, potassium thioglycolate, sodiumthioglycolate, cysteine hydrochloride, ammonium thiolactate,thioglycerin, mercaptoprpionic acid, glycerol thioglycolate anddithiolbutylamine (DTBA). In some embodiments, the reducing agent isselected from the group consisting of ammonium thioglycolate,L-cysteine, glutathione, beta-mercaptoethanol, 2-mercaptoethylamine,DTT, thiolactic acid, TCEP, DTBA, sodium hydrosulfite, and sodiumthiosulfate. In some embodiments, the reducing agent is selected fromthe group consisting of ammonium thioglycolate, L-cysteine, glutathione,and thiolactic acid. In some embodiments, the reducing agent is ammoniumthioglycolate or L-cysteine. In some embodiments, the reducing agent isammonium thioglycolate. In some embodiments, the reducing agent isbeta-mercaptoethanol.

In some embodiments, the reducing agent is a mixture comprising a firstcross-linker comprising a moiety of Formula I. In some embodiments, thereducing agent is a mixture of cyclic disulfides and cyclicthiosulfinate. In some embodiments, the method for treating akeratin-containing material, comprises:

-   -   i) providing a plurality of first monomers, wherein the first        monomer is a keratin-containing material sample comprising a        plurality of disulfide bonds;    -   ii) applying to the keratin-containing material sample for a        period of time a mixture, comprising a first cross-linker,        wherein the first cross-linker comprises a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring, thereby forming a plurality of freethiol groups which react with the first cross-linkers to form aplurality of covalent bonds between the free thiol groups and the firstcross-linkers.

In some embodiments of the methods and polymers disclosed above andbelow, the first cross-linker is selected from the group consisting of1-oxo-1,2-dithiane, 1-oxo-1,2-dithiepane, and 1-oxo-1,2-dithiocane. Insome embodiments, the first cross-linker is 1-oxo-1,2-dithiane.

In some embodiments of the methods and polymers disclosed above andbelow, the ratio of the first monomer to the first cross-linker is fromabout 1:10 to about 10:1. In some embodiments, the ratio of the firstmonomer to the first cross-linker is selected from about 1:10, about1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3,about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about6:1, about 7:1, about 8:1, about 9:1, and about 10:1.

In certain embodiments of the methods disclosed above and below, themethod further comprises contacting the plurality of first cross-linkerswith a plurality of second monomers comprising at least two thiolfunctional groups.

In some embodiments of polymers disclosed above and below, the polymerderived from a first monomer, a first cross-linker, and a secondmonomer.

In some embodiments of the methods and polymers disclosed above andbelow, the second monomer is trithiol compound or a tetrathiol compound,such as trithiocyanuric acid, trimethylolpropanetris(3-mercaptopropionate), and pentaerythritoltetra(3-mercaptopropionate). In some embodiments, the second monomer isselected from the group consisting of trithiocyanuric acid,trimethylolpropane tris(3-mercaptopropionate), and pentaerythritoltetra(3-mercaptopropionate).

In some embodiments of the methods disclosed above and below, the methodfurther comprises contacting the plurality of first cross-linkers with aplurality of second cross-linkers comprising a moiety of Formula I

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring.

In some embodiments of polymers disclosed above and below, the polymerderived from a first monomer, a first cross-linker, and a secondcross-linker.

In some embodiments of the methods and polymers disclosed above andbelow, the plurality of first cross-linkers is different from theplurality of second cross-linkers. In some embodiments, the ratio of theplurality of first cross-linkers to the ratio of the plurality of secondcross-linkers is from about 1:10 to about 10:1. In some embodiments, theratio is selected from about 1:10, about 1:9, about 1:8, about 1:7,about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1,about 9:1, and about 10:1. In some embodiments, the plurality of firstcross-linkers comprise a cyclic disulfide. In some embodiments, firstcross-linkers comprise a cyclic thiosulfinate. In some embodiments, theplurality of second cross-linkers comprise a cyclic disulfide. In someembodiments, the plurality of second cross-linkers comprise a cyclicthiosulfinate.

In another aspect, the present disclosure provides a method of coating asurface of an object, a device, or an assembly, comprising the steps of:

-   -   (a) providing a surface of an object, a device, or an assembly;    -   (b) contacting the surface with a plurality of first molecules        comprising a reactive moiety and a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring;

thereby forming a plurality of covalent bonds between the surface andthe reactive moieties of the first molecule to form a first monolayer.

In some embodiments of the methods of coating a surface, W is S and Y isS or S(O). In some embodiments, the surface is reductive. In someembodiments, the reactive moiety is a thiol or a thiosulfinate.

In some embodiments, the method of coating a surface further comprises

-   -   (c) contacting the monolayer with a second molecule comprising        at least one thiol functional group, thereby forming a first        bilayer, wherein a plurality of covalent bonds are formed        between the S or Se atoms of the first molecule and the free        thiol groups of the second molecule.

In some embodiments of the methods of coating a surface, the secondmolecule is a monothiol compound. In some embodiments, the secondmolecule is selected from the group consisting of(11-mercaptoundecyl)-N,N,N-trimethylammonium bromide,(11-mercaptoundecyl)hexa(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol),1-(11-mercaptoundecyl)imidazole, 1-mercapto-2-propanol,11-(1H-pyrrol-1-yl)undecane-1-thiol, 11-(ferrocenyl)undecanethiol,11-amino-1-undecanethiol hydrochloride, 11-azido-1-undecanethiol,11-mercapto-1-undecanol, 11-mercaptoundecanamide, 11-mercaptoundecanoicacid, 11-mercaptoundecylhydroquinone, 11-mercaptoundecylphos-phonicacid, 12-mercaptododecanoic acid, 16-amino-1-hexadecanethiolhydrochloride, 16-mercaptohexadecanamide, 16-mercaptohexadecanoic acid,3-amino-1-propanethiol hydrochloride, 3-chloro-1-propanethiol,3-mercapto-1-propanol, 3-mercaptopropionic acid, 4-mercapto-1-butanol,6-(ferrocenyl)-hexanethiol, 6-amino-1-hexanethiol hydrochloride,6-mercapto-1-hexanol, 6-mercaptohexanoic acid, 8-amino-1-octanethiolhydrochloride, 8-mercapto-1-octanol, 8-mercaptooctanoic acid,9-mercapto-1-nonanol, triethylene glycol mono-1-mercaptoundecyl ether,1-mercaptosuccinic acid, a peptide having a cysteine residue, a proteinhaving a cysteine residue, cysteamine, 1-thiohexitol, poly(ethyleneglycol) 2-mercaptoethyl ether acetic acid, poly(ethylene glycol) methylether thiol, 1-thioglycerol, 2-naphthalenethiol, biphenyl-4-thiol,3-amino-1,2,4-triazole-5-thiol, 5-(trifluoromethyl)pyridine-2-thiol,1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol, and γ-Glu-Cys.

In some embodiments of the methods of coating a surface, the secondmolecule is selected from the group consisting of a dithiol compound, atrithiol compound, a tetrathiol compound, and a thiomer. In someembodiments, the second molecule is selected from the group consistingof a dithiol compound, a trithiol compound, a tetrathiol compound, ahexathiol compound, and an octathiol compound.

In some embodiments of the methods of coating a surface, the secondmolecule is selected from the group consisting of dithiothreitol (DTT),1,2-ethanedithiol, 1,3-propanedithiol, 1,4-butanedithiol,1,5-pentanedithiol, 1,6-hexanedithiol, 1,7-heptanedithiol,1,8-octanedithiol, 1,9-nonanedithiol, 1,10-decanedithiol,1,11-undecanedithiol, 1,12-dodecanedithiol, 1,13-tridecanedithiol,1,14-tetradecanedithiol, 1,16-hexadecanedithiol, dithiolbutylamine(DTBA), tetra(ethylene glycol) dithiol, hexa(ethylene glycol) dithiol,2-mercaptoethyl ether, 2,2′-thiodiethanethiol,2,2′-(ethylenedioxy)diethanethiol, propane-1,2,3-trithiol,trimethylolpropane tris(2-mercaptoacetate), trimethylolpropanetris(3-mercaptoacetate),3,3′,3″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(propane-1-thiol),4,4′,4″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(butane-1-thiol),pentaerythrityl tetrathiol, pentaerythritoltetrakis(3-mercaptopropionate), a peptide having a disulfide bond, and aprotein having a disulfide bond. In some embodiments, the secondmolecule is a peptide having a disulfide bond or a protein having adisulfide bond. In some embodiments, the second molecule is a proteinhaving a disulfide bond. In some embodiments, the second molecule iscopper/zinc superoxide dismutase (SOD1) or DJ-1, which has beenimplicated in Parkinson's disease. In some embodiments, the secondmolecule is SOD1.

In some embodiments of the methods of coating a surface, the secondmolecule is selected from the group consisting of 4arm-PEG2K-SH,4arm-PEG5K-SH, 4arm-PEG10K-SH, 4arm-PEG20K-SH, 4-arm poly(ethyleneoxide) thiol-terminated, 8arm-PEG10K-SH (hexaglyerol core),8arm-PEG10K-SH (tripentaerythritol core), 8arm-PEG20K-SH (hexaglyerolcore), 8arm-PEG20K-SH (tripentaerythritol core), and 8-arm poly(ethyleneoxide) thiol-terminated.

In certain embodiments of the methods of coating a surface, the secondmolecule is a polyalkylene dithiol, such as polyethylene dithiol,polypropylene dithiol, etc.

In certain embodiments of the methods of coating a surface, the secondmolecule is a trithiol compound or a tetrathiol compound, such astrithiocyanuric acid, trimethylolpropane tris(3-mercaptopropionate), andpentaerythritol tetra(3-mercaptopropionate).

In some embodiments, the method of coating a surface further comprises

-   -   (d) applying an electrical potential, thereby reducing the        covalent bonds, thus forming a second monolayer.

In some embodiments, the method of coating a surface further comprises

-   -   (e) providing an aqueous mixture comprising water and a        plurality of metal ions, thereby forming a complex, comprising a        plurality of metal-chelating groups of the second monolayer        chelated to the plurality of metal ions, thus forming a second        bilayer.

In some embodiments, the method of coating a surface further comprises

-   -   (f) contacting the second bilayer with a plurality of third        molecules comprising a moiety of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring;

thereby forming a trilayer, wherein the plurality of third molecules arechelated to the plurality of metal ions.

In another aspect, provided herein is a composition, wherein thecomposition comprises a substrate and a coating material, wherein thecoating material comprises a first monolayer comprising a plurality ofmoieties of Formula I:

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring; and

the coating material is covalently bonded to the substrate.

In some embodiments of the composition, the first monolayer comprises aplurality of moieties selected from the group consisting of

In some embodiments, the first monolayer comprises a plurality ofmoieties selected from the group consisting of

In some embodiments of the composition, the coating material comprises afirst bilayer comprising a polymer derived from the moieties of FormulaI and a plurality of first monomers comprising at least one thiolfunctional group, wherein a plurality of covalent bonds are formedbetween the S or Se atoms of the plurality of moieties and the freethiol groups of the first monomer.

In some embodiments of the composition, the coating material comprises asecond monolayer comprising a reduced form of the plurality of moietiesof Formula I.

In some embodiments of the composition, the coating material comprises asecond bilayer comprising a plurality of metal-chelating groups of thesecond monolayer chelated to a plurality of metal ions.

In some embodiments of the methods of coating or coating compositions,the plurality of metal-chelating groups are S atoms. In someembodiments, the plurality of metal-chelating groups are Se atoms.

In some embodiments of the methods of coating or coating compositions,the plurality of metal ions are a plurality of metal cations.

In some embodiments of the methods of coating or coating compositions,the metal cation has a charge of +1. In some embodiments, the metalcation is a cation of Ag or Au.

In some embodiments of the methods of coating or coating compositions,the metal cation has a charge of +2. In some embodiments, the metalcation is a cation of Ca, Cd, Co, Cr, Cu, Er, Fe, Hg, Mg, Mn, Nb, Ni,Pb, Pd, Sc, Sn, Sr, V, or Zn. In some embodiments, the metal cation is acation of Cd, Co, Cr, Cu, Fe, Hg, Mn, Nb, Ni, Pb, Pd, Sn, Sr, V, or Zn.

In some embodiments of the methods of coating or coating compositions,the metal cation has a charge of +3. In some embodiments, the metalcation is a cation of Au, Ce, Dy, Er, Eu, Fe, Gd, Ho, La, Lu, Nb, Nd,Pm, Pr, Sm, Tb, Tm, or Yb. In some embodiments, the metal cation is acation of Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pm, Pr, Sm, Tb, Tm, or Yb.In some embodiments, the metal cation is a cation of Au, Fe, or Gd. Insome embodiments, the metal cation is a cation of Au or Fe.

In some embodiments of the composition, the coating material comprises atrilayer further comprising a plurality of third molecules chelated tothe plurality of metal ions, wherein the third molecules comprise amoiety of Formula I

wherein

W is independently selected from the group consisting of S and Se;

Y is independently selected from the group consisting of S, Se, S(O),Se(O), S(O)₂ and Se(O)₂; and

X together with W and Y forms a substituted or unsubstituted3-10-membered heterocyclic ring.

In summary, cyclic disulfide and cyclic diselenide reactivity, includingreversible binding to lone thiols, is predictable and highly tunable.Cyclic thiosulfinate, cyclic thioselenoxide and cyclic selenosulfinatecross-linkers have potential as:

1) A less toxic alternative to Cys specific diene cross-linkers andphenylarsine oxide cross-linkers, which can both react withmonothiols,³³

2) Probes for proteinaceous Cys-dithiolates, which perform essential invivo functions and often serve as metal and metallocofactorligands,³⁴⁻³⁶

3) Inter-functional group distance measurement tools,

4) Biocompatible templates for higher order structures in polymersynthesis, and

5) Cellular thiol pair cross-linkers.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry described herein, arethose well-known and commonly used in the art.

Throughout the specification and the claims, unless the context requiresotherwise, the words “comprise” and “include” and variations such as“comprising” and “including” will be understood to imply the inclusionof a stated integer or group of integers, but not the exclusion of anyother integer or group of integers.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkyC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group, having an oxygen attachedthereto. Representative alkoxy groups include methoxy, trifluoromethoxy,ethoxy, propoxy, tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Typically, a straight chainedor branched alkenyl group has from 1 to about 20 carbon atoms,preferably from 1 to about 10 unless otherwise defined. Suchsubstituents may occur on one or more carbons that are included or notincluded in one or more double bonds. Moreover, such substituentsinclude all those contemplated for alkyl groups, as discussed below,except where stability is prohibitive. For example, substitution ofalkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, orheteroaryl groups is contemplated.

An “alkyl” group or “alkane” is a straight chained or branchednon-aromatic hydrocarbon which is completely saturated. Typically, astraight chained or branched alkyl group has from 1 to about 20 carbonatoms, preferably from 1 to about 10 unless otherwise defined. In someembodiments, the alkyl group has from 1 to 8 carbon atoms, from 1 to 6carbon atoms, from 1 to 4 carbon atoms, or from 1 to 3 carbon atoms.Examples of straight chained and branched alkyl groups include methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl,hexyl, pentyl and octyl.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more substitutablecarbons of the hydrocarbon backbone. Such substituents, if not otherwisespecified, can include, for example, a halogen (e.g., fluoro), ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxy, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. In preferred embodiments, thesubstituents on substituted alkyls are selected from C₁₋₆ alkyl, C₃₋₆cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferredembodiments, the substituents on substituted alkyls are selected fromfluoro, carbonyl, cyano, or hydroxyl. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed below. Cycloalkyls can be further substituted with alkyls,alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,—CF₃, —CN, and the like.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y) alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups. Preferred haloalkyl groups includetrifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, andpentafluoroethyl. C₀ alkyl indicates a hydrogen where the group is in aterminal position, a bond if internal. The terms “C_(2-y) alkenyl” and“C_(2-y) alkynyl” refer to substituted or unsubstituted unsaturatedaliphatic groups analogous in length and possible substitution to thealkyls described above, but that contain at least one double or triplebond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS-.

The term “arylthio”, as used herein, refers to a thiol group substitutedwith an alkyl group and may be represented by the general formulaarylS-.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Typically, a straight chainedor branched alkynyl group has from 1 to about 20 carbon atoms,preferably from 1 to about 10 unless otherwise defined. Suchsubstituents may occur on one or more carbons that are included or notincluded in one or more triple bonds. Moreover, such substituentsinclude all those contemplated for alkyl groups, as discussed above,except where stability is prohibitive. For example, substitution ofalkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, orheteroaryl groups is contemplated.

The term “amide”, as used herein, refers to a group

wherein each R^(A) independently represent a hydrogen or hydrocarbylgroup, or two R^(A) are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R^(A) independently represents a hydrogen or a hydrocarbylgroup, or two R^(A)are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 6- or 20-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein each R^(A) independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group, or both R^(A) taken together with theintervening atom(s) complete a heterocycle having from 4 to 8 atoms inthe ring structure.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.Preferably, a carbocylic group has from 3 to 20 carbon atoms. The termcarbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

A “cycloalkyl” group is a cyclic hydrocarbon which is completelysaturated. “Cycloalkyl” includes monocyclic and bicyclic rings.Preferably, a cycloalkyl group has from 3 to 20 carbon atoms. Typically,a monocyclic cycloalkyl group has from 3 to about 10 carbon atoms, moretypically 3 to 8 carbon atoms unless otherwise defined. The second ringof a bicyclic cycloalkyl may be selected from saturated, unsaturated andaromatic rings. Cycloalkyl includes bicyclic molecules in which one, twoor three or more atoms are shared between the two rings. The term “fusedcycloalkyl” refers to a bicyclic cycloalkyl in which each of the ringsshares two adjacent atoms with the other ring. The second ring of afused bicyclic cycloalkyl may be selected from saturated, unsaturatedand aromatic rings. A “cycloalkenyl” group is a cyclic hydrocarboncontaining one or more double bonds.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate”, as used herein, refers to a group —OCO₂—R^(A),wherein R^(A) represents a hydrocarbyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR^(A) whereinR^(A) represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical.

Examples of ethers include, but are not limited to,heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include“alkoxyalkyl” groups, which may be represented by the general formulaalkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms and at least one heteroatom, whereinno two heteroatoms are adjacent.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to20-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 20-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms.

The terms “heterocyclyl” and “heterocyclic” also include polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.

Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom, wherein that carbon atom does not have a ═O or ═Ssubstituent. Hydrocarbyls may optionally include heteroatoms.Hydrocarbyl groups include, but are not limited to, alkyl, alkenyl,alkynyl, alkoxyalkyl, aminoalkyl, aralkyl, aryl, aralkyl, carbocyclyl,cycloalkyl, carbocyclylalkyl, heteroaralkyl, heteroaryl groups bondedthrough a carbon atom, heterocyclyl groups bonded through a carbon atom,heterocyclylakyl, or hydroxyalkyl. Thus, groups like methyl,ethoxyethyl, 2-pyridyl, and trifluoromethyl are hydrocarbyl groups, butsubstituents such as acetyl (which has a ═O substituent on the linkingcarbon) and ethoxy (which is linked through oxygen, not carbon) are not.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are six or fewer non-hydrogen atoms in thesubstituent. A “lower alkyl”, for example, refers to an alkyl group thatcontains six or fewer carbon atoms. In some embodiments, the alkyl grouphas from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, or from 1 to 3carbon atoms. In certain embodiments, acyl, acyloxy, alkyl, alkenyl,alkynyl, or alkoxy substituents defined herein are respectively loweracyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or loweralkoxy, whether they appear alone or in combination with othersubstituents, such as in the recitations hydroxyalkyl and aralkyl (inwhich case, for example, the atoms within the aryl group are not countedwhen counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

In the phrase “poly(meta-phenylene oxides)”, the term “phenylene” refersinclusively to 6-membered aryl or 6-membered heteroaryl moieties.Exemplary poly(meta-phenylene oxides) are described in the first throughtwentieth aspects of the present disclosure.

The term “silyl” refers to a silicon moiety with three hydrocarbylmoieties attached thereto.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.Moieties that may be substituted can include any appropriatesubstituents described herein, for example, acyl, acylamino, acyloxy,alkoxy, alkoxyalkyl, alkenyl, alkyl, alkylamino, alkylthio, arylthio,alkynyl, amide, amino, aminoalkyl, aralkyl, carbamate, carbocyclyl,cycloalkyl, carbocyclylalkyl, carbonate, ester, ether, heteroaralkyl,heterocyclyl, heterocyclylalkyl, hydrocarbyl, silyl, sulfone, orthioether. As used herein, the term “substituted” is contemplated toinclude all permissible substituents of organic compounds. In a broadaspect, the permissible substituents include acyclic and cyclic,branched and unbranched, carbocyclic and heterocyclic, aromatic andnon-aromatic substituents of organic compounds. The permissiblesubstituents can be one or more and the same or different forappropriate organic compounds. For purposes of this invention, theheteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. Substituents can include anysubstituents described herein, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. In preferred embodiments, the substituents onsubstituted alkyls are selected from C₁₋₆ alkyl, C₃₋₆ cycloalkyl,halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments,the substituents on substituted alkyls are selected from fluoro,carbonyl, cyano, or hydroxyl. It will be understood by those skilled inthe art that substituents can themselves be substituted, if appropriate.Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group—S(O)₂—R^(A), wherein R^(A) represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

Examples

Materials and Instrumentation

1,4-butanedithiol used in the synthesis of both 1,2-dithiane and1,2-dithiane-1-oxide was purchased through TCI America (Portland, Oreg.,USA). All buffer components and solvents (including ESI solvents) usedin the kinetic assay experiments were purchased through FisherScientific (Hampton, N.H., USA) except formic acid which was purchasedthrough Sigma-Aldrich (St. Louis, Mo., USA). Incubation of samples wasdone in an Eppendorf Thermomixer R (Hauppauge, N.Y., USA). All cellculture ingredients were purchased through ATCC (Manassas, Va., USA).Cells were incubated in a ThermoFisher Isotemp incubator (Waltham,Mass., USA). SDS Page was performed using a MiniProtean electrophoresischamber (Hep G2 experiments) and Criterion electrophoresis chamber (HeLaexperiments) from Bio Rad (Hercules, Calif., USA). Cu/Zn SOD polyclonalantibody was purchased through Enzo (Farmingdale, N.Y., USA). PierceSecondary antibody was purchased through Thermo Scientific (Waltham,Mass., USA). Membranes were imagined on a Bio Rad ChemiDoc MP (Hercules,Calif., USA). Mass spectra for kinetic assays was acquired on a BrukerSolarix XR FT-ICR mass spectrometer with an electrospray ionizationsource and a 9.4 Tesla magnet. Acquired data was analyzed and processedusing Bruker Daltronics DataAnalysis 4.4.102 software (Billerica, Mass.,USA). ¹H NMR spectra were recorded at ambient temperature on a VarianMercury NMR spectrometer (Palo Alto, Calif., USA) operating at 400 MHzin the solvent indicated with the signal of the residual solvent (CHCl₃δ 7.26 ppm) as internal standard. ¹³C NMR spectra were recorded with ¹Hdecoupled observation at ambient temperature on a Varian NMRspectrometer operating at 100 MHz in the solvent indicated with thesignal of the residual solvent (CHCl₃ δ 77.16 ppm) as internal standard.Data are reported as follows: chemical shift, multiplicity (m=multiplet,dt=doublet of triplets, td=triplet of doublets, dtt=doublet of tripletof triplets), integration and coupling constant (Hz). Thin-layerchromatography (TLC) was performed with silica gel 60 F254 pre-coatedplates and visualized with exposure to UV light (254 nm) or by potassiumpermanganate stain (KMnO₄) followed by heating. Phenylarsine oxide waspurchased through Sigma-Aldrich (St. Louis, Mo., USA). SOD1 (WT andC111S) yeast expression vectors were kindly provided by P.J Hart (TheUniversity of Texas Health Science Center). Both phenyl-sepharosehydrophobic interaction chromatography column and Mono Q 10/100 anionexchange chromatography column was purchased through GE Life Sciences(Piscataway, N.J., USA) and protein purification was performed on anÄkta Fast Protein Liquid Chromatography (FPLC) (Piscataway, N.J., USA).LC-MS data was collected on a Bruker HCT Ultra ion trap (Billerica,Mass., USA).

Methods

Synthesis of 1,2-dithiane

Preparation of 1,2-dithiane was adapted from a known literatureprocedure.³⁷ Silica gel (40-60 μm particle size, 60 Å pore size, 41 g)was added to a round bottom flask and distilled water (102 mL) was addedslowly with rigorous stirring until a uniform suspension had formed.Dichloromethane (200 mL) and 1,4-butanedithiol (2.00 g, 16.4 mmol, 1equiv) were added to the suspension while stirring. A solution of Br₂(2.88 g, 18 mmol, 1.10 equiv) in dichloromethane (16 mL) was addeddropwise to the off-white suspension while stirring vigorously. Thereaction mixture was stirred for 5 minutes, and reaction completion wasconfirmed by TLC analysis. The reaction mixture was filtered over celiteinto a flask containing a stirred solution of 1.25 M NaOH (12 mL). Thecolorless organic phase was removed, washed with distilled water (3×50mL) and dried over sodium sulfate. The solvent was removed under reducedpressure and the crude product crystallized from hexanes at −20° C. toyield 1,2-dithiane as a white crystalline solid (1.63 g, 83%).R_(f)=0.80 (5:1 hexanes:EtOAc); ¹H NMR (400 MHz, CDCl₃, δ): 1.97 (bs,4H), 2.85 (bs, 4H); ¹³C NMR (100 MHz, CDCl₃, δ): 27.9, 33.5. mp 29-32°C. (lit. 28-30° C.).³⁷

Synthesis of 1,2-dithiane-1-oxide

Preparation of 1,2-dithiane-1-oxide was adapted from a known literatureprocedure.³⁷ A solution of sodium periodate (843 mg, 3.94 mmol, 1.10equiv) in water (64 mL) was added dropwise to a stirred solution of1,2-dithiane (431 mg, 3.58 mmol, 1 equiv) in methanol (193 mL) at 0° C.The reaction mixture was stirred 16 h, and reaction completion wasconfirmed by TLC analysis. The white slurry warmed to room temperature,filtered over celite, and the filtrate was concentrated under reducedpressure. The remaining solution was diluted with chloroform (40 mL) andtransferred to a separatory funnel. A small amount of solid NaCl wasadded and the aqueous layer was extracted with CHCl₃ (3×40 mL). Thecombined organic layer was dried over sodium sulfate, the solventremoved under reduced pressure, and purified by flash columnchromatography on silica gel with 2% MeOH/CH₂Cl₂ yielded1,2-dithiane-1-oxide as a colorless solid (214 mg, 44%). R_(f)=0.33 (2%MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃, δ): 1.85-1.88 (m, 1H), 1.96-2.06(dtt, J=13.7, 12.7, 3.0 Hz, 1H), 2.11-2.15 (m, 1H), 2.62-2.71 (m, 2H),3.04-3.12 (dt, J=13.1, 3.0 Hz, 1H), 3.18-3.23 (td, J=13.3, 3.6 Hz, 1H),3.62-3.70 (ddd, J=14.5, 12.0, 2.5 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃, δ):15.3, 23.5, 25.8, 51.9. HRMS-ESI (m z): [M+H]⁺ calcd for C₄H₈OS₂,137.00893 Da; found 137.00893 Da. mp 83-86° C. (lit. 85° C.).³⁷

Synthesis of 1,2-dithiepane

Preparation of 1,2-dithiepane was adapted from a known literatureprocedure.³⁷ Silica gel (40-60 μm particle size, 60 Å pore size, 46 g)was added to a round bottom flask and distilled water (23 mL) was addedslowly with rigorous stirring until a uniform suspension had formed.Dichloromethane (230 mL) and 1,5-pentanedithiol (3.00 g, 22.01 mmol, 1equiv) were added to the suspension while stirring. A solution of Br₂(3.87 g, 24.22 mmol, 1.10 equiv) in dichloromethane (23 mL) was addeddropwise to the off-white suspension while stirring vigorously. Thereaction mixture was stirred for 5 minutes, and reaction completion wasconfirmed by TLC analysis. The reaction mixture was filtered over celiteinto a flask containing a stirred solution of 1.25 M NaOH (110 mL). Thecolorless organic phase was removed, washed with distilled water (3×70mL) and dried over sodium sulfate. The solvent was removed under reducedpressure and the crude product purified by flash column chromatographyon silica gel with 20:1 Hex/EtOAc yielded 1,2-dithiepane as a clearliquid (2.83 g, 96%). R_(f)=0.82 (9:1 hexanes:EtOAc); ¹H NMR (400 MHz,CDCl₃, δ): 1.75-1.79 (m, 2H), 2.00-2.05 (m, 4H), 2.82-2.84 (t, J=6.3,4H); ¹³C NMR (100 MHz, CDCl₃, δ): 26.0, 30.0, 39.2.

Synthesis of 1,2-dithiepane-1-oxide

Preparation of 1,2-dithiepane was adapted from a known literatureprocedure for the oxidation of disulfides.³⁸ mCPBA (73%, 214 mg, 0.909mmol, 1.07 equiv) was added at 0° C. to a solution of 1,2-dithiepane(114 mg, 0.849 mmol, 1 equiv) in anhydrous dichloromethane (5.5 mL). Thesolution was stirred in an ice bath for 1 h, then sodium carbonate (1 g)was added and stirred for 30 min at 0° C. The solution was filtered overa celite pad and magnesium sulfate, solvent was removed under reducedpressure and the crude product purified by flash column chromatographyon silica gel with 5:1 Hex/EtOAc yielded 1,2-dithiepane-1-oxide as aclear liquid (97 mg, 76%). R_(f)=0.19 (5:1 hexanes:EtOAc); ¹H NMR (400MHz, CDCl₃, δ): 1.73-1.81 (m, 1H), 1.91-2.02 (m, 4H), 2.09-2.16 (m, 1H),2.77-2.86 (m, 2H), 3.36-3.41 (dd, J=6.5, 13.8 Hz, 1H), 3.50-3.57 (m,1H); ¹³C NMR (100 MHz, CDCl₃, δ): 18.02, 22.31, 26.77, 28.46, 60.56.HRMS-ESI (m z): [M+H]⁺ calcd for C₅H₁₀OS₂, 151.024584 Da; found151.02498 Da.

Synthesis of β-Lipoic Acid

Preparation of β-lipoic acid was adapted from a known literatureprocedure.³⁹ Aqueous hydrogen peroxide (0.841 mL, 35% in H2O, 9.77 mmol,2 equiv) was added to a solution of DL-thioctic acid (α-lipoic acid,1.01 g, 4.89 mmol, 1 equiv) in acetone (2.5 mL) and allowed to stir for24 h. Solvent was removed under reduced pressure, diluted withdichloromethane (25 mL) and then added to brine (50 mL). The aqueouslayer was extracted with dichloromethane (3×25 mL) and dried over sodiumsulfate. The solvent was removed under reduced pressure and purified byflash column chromatography on silica gel with 3% MeOH/CH₂Cl₂, 0.1% AcOHyielded beta-lipoic acid as a colorless oil (418 mg, 39%). R_(f)=0.46(8% MeOH/CH₂Cl₂, 0.1% AcOH). ¹H-NMR spectrum and ¹³C-NMR data matchedthose reported by others³⁹⁻⁴⁰ for a mixture of all four stereo- andregioisomers: the H-NMR and ¹³C-NMR chemical shifts have been exactlyobserved as reported by 2D techniques performed by Müller et al.⁴⁰HRMS-ESI (m/z): [M+H]+calcd for C4H14O3S2, 223.04571 Da; found 223.04626Da.

Kinetics of 1,2-dithaine-1-oxide and 1,2-dithaine cross-linking

To test the cross-linking activity of cyclic disulfides and cyclicthiosulfinates, 1,2-dithiane and 1,2-dithiane-1-oxide were synthesizedand incubated with SOD1, a homodimeric protein containing a solventaccessible thiol pair (Cy_(111A+B), 8 Å apart) on adjacent subunits. Thereaction was monitored using a mass spectrometry (MS) assay that uses acombination of increased voltage within the region of hypersonic gasexpansion and brief treatment with 10% formic acid to create exclusivelymonomeric SOD1 or covalently cross-linked SOD1 dimer. Formic acid alsoquenches the reaction, transforming any reactive thiolates to unreactivethiols. As a result, if no reaction occurs, only apo SOD1 monomer isdetected (FIG. 2, top). Consistent with our hypothesized mechanism: 1)1,2-dithiane-1-oxide, but not 1,2-dithiane, resulted in rapid andcomplete dithiolate cross-linking (half-life ˜2-3 min) of SOD1 (FIG. 2,middle and FIG. 3); 2) no binding to single Cys residues (SOD1 has freeCys₁₁₁ and Cys₆) was observed in any sample with either compound; 3) Nocross-linking was observed without the loss of the oxygen from the S-oxoof 1,2-dithiane-1-oxide; 4) No cross-linking was observed whenincubating 1,2-dithiane-1-oxide with C₁₁₁S SOD1 (FIG. 4). Givensufficient time for thiolates to be oxidized to sulfenic acid (whichoccurs on the order of days-weeks), even 1,2-dithiane was expected tocross-link SOD1. After 72 h of incubation with 1,2-dithiane, 11% of SOD1had formed the expected covalent dimer (FIG. 5). Comparable results wereobserved from the incubation of SOD1 with 1,2-dithiepane and1,2-dithiepane-1-oxide (FIG. 13). Comparable results were observed fromthe incubation of SOD1 with 1,2-dithiocane and 1-oxo-1,2-dithiocane(FIG. 15).

Kinetic studies of the cross-linking efficiency of 1,2-dithiane comparedto 1,2-dithiane-1-oxide were performed in tandem. 50 μM human WT SOD1 in10 mM ammonium acetate pH 7.4 was incubated with 1000 μM 1,2-dithiane or1,2-dithiane-1-oxide (20× excess) at 37° C. Human WT SOD1 was expressedin yeast. Both 1,2-dithiane and 1,2-dithiane-1-oxide were dissolved in100% HPLC grade methanol to a stock concentration of 25 mM. Dilution inHPLC-Grade water to 1 mM cyclic disulfide was performed prior toexperimentation. As a negative control, 50 μM human WT SOD1 wasincubated at 37° C. with 4% HPLC-grade methanol. At every time point, 1μL of each sample was removed from their respective reaction vial foranalysis. Samples were briefly (30 seconds) incubated at roomtemperature with 10% formic acid to remove metals from SOD1 and quenchcross-linking reaction prior to mass spectrometry analysis. Samples werethen diluted to 1 μM SOD1 in 50:50 acetonitrile:water, 0.1% FA andanalyzed by direct infusion into a Bruker SolariX XR FT-ICR massspectrometer. The percent dimer formation was calculated by comparingthe relative dimer (31,808 Da) and monomer (15,844 Da) MaxEntdeconvoluted peak heights (Dimer/(Dimer+Monomer)). Identical methodswere used in the kinetics of α-lipoic acid and β-lipoic acid. Kineticsof 1,2-dithiepane vs. 1,2-dithiepane-1-oxide were performed at a 1:1ratio of SOD1 dimer:cross-linker.

Expression and Purification of hSOD1 (WT and C111S)

Expression and purification of WT SOD1 and C111S SOD1 was carried out aspreviously published.⁴¹⁻⁴³ Briefly, human SOD1 cDNA cloned into theyeast expression vector YEp-351 was transformed into EGy118ΔSOD1 yeastand grown at 30° C. for 36-48 hours. Cultures were pelleted, lysed using0.5 mm glass beads and a blender, and subjected to a 60% ammoniumsulfate cut on ice. After ammonium sulfate precipitation, the sample waspelleted and the supernatant was diluted with ˜0.19 volumes buffer (50mM sodium phosphate, 150 mM sodium chloride, 0.1 M EDTA, 0.25 mM DTT, pH7.0) to a final concentration of 2.0 M ammonium sulfate. The sample wasthen purified using a phenyl-Sepharose hydrophobic interactionchromatography column with a 300 mL linearly decreasing salt gradientfrom a high salt buffer (2.0 M ammonium sulfate, 50 mM sodium phosphate,150 mM sodium chloride, 0.1 M EDTA, 0.25 mM DTT, pH 7.0) to a low saltbuffer (50 mM sodium phosphate, 150 mM sodium chloride, 0.1 M EDTA, 0.25mM DTT, pH 7.0). Samples containing SOD1 typically elute between 1.6-1.1M ammonium sulfate, which was confirmed using gel electrophoresis. SOD1containing fractions were pooled and exchanged to a 10 mM Tris (pH 8.0)buffer. The protein was then loaded onto a Mono Q 10/100 anion exchangechromatography column and eluted using a 200 mL linearly increasing saltgradient from a low salt buffer (10 mM Tris, pH 8.0) to a high saltbuffer (10 mM Tris, pH 8.0, 1 M sodium chloride). The gradient is runfrom 0-30% 10 mM Tris, pH 8.0, 1 M sodium chloride and SOD1 elutedbetween 5-12% 10 mM Tris, pH 8.0, 1 M sodium chloride. SOD1 fractionsfrom anion exchange were confirmed using gel electrophoresis. Final SOD1containing fractions were pooled together, washed three times with 10 mMammonium bicarbonate in a Millipore amicon centrifugal filter, andbuffer exchanged into 10 mM ammonium acetate pH 7.4. Protein was storedat −80° C. until use.

Localization of Cross-Link Using Point Mutated C111S SOD1

1,2-Dithiane-1-oxide was initially dissolved in 100% DMSO. Recombinanthuman WT SOD1 and C111S SOD1 purified from yeast (10 μM) were incubatedovernight at room temperature with 1,2-dithiane-1-oxide at 100 μM in0.1% DMSO after dilution. Aliquots from each of the SOD1 incubationswere individually diluted ten-fold with H₂O containing 0.1% formic acidand analyzed using reversed phase C18 LC-MS on a Bruker HCT Ultra iontrap. The resulting data were processed using DataAnalysis 3.4 (BrukerDaltonics). Mass spectra were averaged across the retention timescorresponding to when SOD1 eluted and were deconvoluted to determine themolecular weight of the uncharged species. Note, the acidic conditionsemployed during liquid chromatography and the relatively harshelectrospray ionization process resulted in loss of native metals andnative dimer dissociation.

In Vitro Glutathione Competition Assay

Purified WT SOD1 was diluted to 10 μM in 10 mM ammonium acetate, pH 7.4.Protein was incubated with freshly prepared 100 μM 1,2-dithiane-1-oxidein 5% methanol and 1000 μM reduced glutathione (in water) at 37° C. forgiven time periods. Samples were extracted, briefly (10 s) with 10%formic acid to and analyzed at 0 min, 1 min, 10 min, 100 min, and 1000mins. Control samples included: 1) 10 μM SOD1 2) 10 μM SOD1+100 μM1,2-dithiane-1-oxide 3) 10 μM SOD1+1000 μM reduced glutathione. Sampleswere briefly (30 seconds) incubated at room temperature with 10% formicacid to remove metals from SOD1 prior to mass spectrometry analysis.Samples were then diluted to 1 μM SOD1 in 50:50 acetonitrile:water, 0.1%FA and analyzed by direct infusion into a Bruker Solarix XR FT-ICR massspectrometer. The percent dimer formation was calculated by comparingthe relative dimer (31,808 Da) and monomer (15,844 Da) MaxEntdeconvoluted peak heights (Dimer/(Dimer+Monomer))

In Vitro DTT Competition Assay

Purified WT SOD1 was diluted to 50 μM in 10 mM ammonium acetate, pH 7.4.2 μL of 50 μM SOD1 solution was incubated with 1 μL of 2 mM1,2-dithiane-1-oxide and 1 μL of various concentrations of DTT. DTTsolutions were made in 100% HPLC grade H₂O. DTT solution concentrationswere 1 mM, 2 mM and 4 mM. Samples were left to incubate at 37° C. for 24h. Samples were then diluted to 1 μM SOD1 in 50:50 acetonitrile:water,0.1% FA and analyzed by direct infusion into a Bruker Solarix XR FT-ICRmass spectrometer

Cell Culture and 1,2-Dithiane-1-Oxide/1,2-Dithiane Dosing and α-LipoicAcid/β-Lipoic Acid in Hep-G2 Cells

To demonstrate the utility of 1,2-dithaine-1-oxide as a cellpenetrating, dithiol pair cross-linker, the cross-linking reaction wasexamined both in two widely used human cell lines (Hep G2 and HeLa), andwith purified SOD1 in the presence of competing reduced glutathione orDTT. Glutathione is present in human cells in concentrations up to 7 mM;Hep G2 and HeLa cells both contain approximately 5 mM glutathione.³⁰ HepG2 cells (FIG. 2, bottom) and HeLa cells (FIG. 6) incubated with variousconcentrations of 1,2-dithaine-1-oxide for 30 min showed an EC₅₀ of ˜5μM in western blots, confirming that cellular conditions do not prohibitcross-linking. Cell viability was not affected by 1,2-dithiane-1-oxideconcentrations that were 50-fold higher than the EC₅₀, and the LC₅₀ of1,2-dithiane-1-oxide was approximately 200-fold greater than its EC₅₀(FIG. 7). Consistent with the cellular studies, cross-linking ofpurified SOD proceeded to completion in the presence of 10:1 ratio ofglutathione:1,2-dithiane-1-oxide (FIG. 10), and even in the presence ofequimolar concentrations of the reducing agent dithiothreitol (DTT)(FIG. 11). The rate of cross-linking was decreased in the presence ofcompeting reductants, presumably due to reversible thiolate-disulfideinterchange between reductants and 1,2-dithiane-1-oxide (no glutathionylor DTT adducts with 1,2-dithiane-1-oxide or SOD1 were observed). Theseresults confirm the utility of these cross-linkers even in presence ofmodest amounts of additional reducing agents.

Cyclic disulfides³¹ and their derivatives (e.g., dithiolene thiones)²⁴have been used therapeutically and many of their targets are known.However, the binding mechanism of these drugs, including that of thenutritional supplement and diabetic complication treatment, α-lipoicacid (ALA), have not been characterized.²⁵ To broaden the applicabilityof cyclic disulfide mediated cross-linking and explore a potential modeof action, ALA was purchased and β-lipoic acid (BLA), which was observedin mass spectrometry assays of ALA nutritional supplements, wassynthesized and assayed as above. Compared to ALA, BLA cross-linked SOD1in cells and cross-linked 30% more SOD1 in vitro (FIG. 8 and FIG. 9).Notably, the terminal carboxylic acid on ALA and BLA presents anopportunity for functionalization.³²

Hep G2 cells were cultures in DMEM with 10% fetal bovine serum andpenicillin/streptomycin in 96 well Costar® Corning CellBIND plate with5% CO₂ at 37° C. The cells were cultured to monolayer confluency. 20 mM,2 mM, 200 μM, 20 μM, and 2 μM stocks of 1,2-dithiane and1,2-dithiane-1-oxide were prepared in 100% DMSO. Stocks were dilutedtwenty-fold in 1×PBS (final DMSO 5%). Cells were treated with 200 uL ofcompound and incubated for 30 minutes at 37° C. 5% CO₂. Cells werewashed with 1×PBS and 20 μL of 6× nonreducing sample buffer was added toeach well. The 96 well plate was heated to 90° C. for 10 minutes.Samplers were spun at 14000 RPM for 5 minutes in a Beckman CoulterMicrofuge®18 centrifuge. Samples were run using a Bio Rad Mini-Proteanelectrophoresis chamber on 12% precast TGX polyacrylamide gels at 150V.After separation was complete, gels were extracted from the cassettesand incubated in an in-house transfer buffer for 10 minutes are 90° C.to ensure thorough transfer of protein bands and thorough binding ofantibody (25 mM Tris, 192 mM glycine, 10 mM 2-mercaptoethanol, 0.1%SDS). Transfer was performed with a Bio Rad Trans-blot turbo transfersystem using a trans-clot turbo transfer pack. Membranes were dried,blocked with 5% milk for 2 h at room temperature. Membranes were thenprobed with primary antibody, anti-SOD1 antibody SOD100 overnight at 4°C. Membranes are incubated with HRP-labeled secondary antibodies andvisualized using ECL Western Blotting Substrate and imaged using theChemiDoc MP.

Cell Culture and 1,2-Dithaine-1-Oxide Dosing Assay in HeLa Cells

HeLa cells were cultured in DMEM with 10% fetal bovine serum andpenicillin/streptomycin in 24 well Costar® Corning CellBIND plate with5% CO₂ at 37° C. The cells were cultured to monolayer confluency. A 100mM concentration of 1,2-dithiane-1-oxide was made by dissolving in DMSO.The 100 mM solution diluted to various concentrations with 1×PBSsolution. 1 mL of solutions was added to the HeLa cells in the 24 wellplate. 1,2-Dithiane-1-oxide was allowed to interact with the cells for 1h. Cells were washed 2× with 1 mL PBS. After removing the second PBSwash, the cells were then treated with 6× non-reducing SDS sample bufferand collected. The collected cell lysate was divided into two parts. Onehalf was treated with 5% β-ME, to remove bound cyclic disulfides, asnegative controls. The other half was treated with equal amount ofmilliQ water. All the cell lysate samples were then heated at 80° C. for10 minutes in an Eppendorf Thermomixer. The samples were spun at 14000rpm for 5 minutes in Beckman Coulter Microfuge centrifuge. HeLa cellextracts were separated on 12% SDS-polyacrylamide Tris HCl gels run at80 V at room temperature. Proteins were transferred to nitrocellulosemembranes for Western blotting. Monomer and covalently-linked dimer SOD1were detected using the rabbit polyclonal anti-SOD1 antibody SOD100.Membranes are incubated with HRP-labeled secondary antibodies andvisualized using ECL Western Blotting Substrate and imaged using theChemiDoc MP.

Cell Culture and 1,2-dithiane-1-oxide Cell Viability Assay

Human immortalized neuroblastoma cells (SH-SY5Y) were purchased fromATCC (Manassas, Va., USA). Cells were grown in Eagle's Minimum EssentialMedium (EMEM) supplemented with 10% fetal bovine serum at 37° C. and 5%CO₂ to >70% confluency. Cells were treated with 200 μL of variousconcentrations of 1,2-dithiane-1-oxide in 2% DMSO (250 μM, 500 μM, 750μM, and 1000 μM) and allowed to incubate for 24 h in a 96 well plate(Corning Inc., Corning, N.Y., USA). Following incubation with1,2-dithaine-1-oxide, 22 μL of PrestoBlue® was added to each well andallowed to incubate for 10 min at 37° C. Excitation/emission wasmeasured at 560/590 (in nm) using an Infinite® 200 plate reader (Tecan,Mannedorf, Switzerland). Cell viability was quantified relative tountreated cells.

Computational Methods

All computations were carried out with Gaussian 09, Rev E.01.⁴⁴ Thereported stationary points were optimized using the M06-2X⁴⁵ densityfunctional at the DFT level of theory. The Gaussian 09 default ultrafineintegration grid and the polarizable continuum model (PCM) using theintegral equation formalism variant (IEF-PCM)⁴⁶⁻⁴⁸ using parameters forwater were utilized. Each stationary point was subjected to avibrational analysis and all transition structures have exactly onenegative frequency, while all other stationary points have allfrequencies greater than 0 indicating local minima. A single pointenergy calculation was then performed on the 6-31+G(d,p)-optimizedgeometries at the DFT level of theory with ultrafine grid and PCM modelas mentioned earlier. Single point energy were performed using the with6-311+G(d,p) basis set. The total energy was calculated by adding thefree energy correction and the single point energy.

The parameters for the transition structure conformational search wereidentical for the minima, but the forming S—S bond was constrained at2.4 Angstroms for the cyclic disulfide ring opening step (TS5->2a andTS1->2b) and 2.3 Angstroms for the thiolate/sulfenic acid condensationreaction (TS(3a,b->4)). Each constrained transition state searchproduced 10 structures within 12 kcal/mol (according to OPLS2005) whichwere subjected to a transition state search. The reported structures inFIG. 1 are the lowest energy transition structures for eachtransformation.

QM Calculations are Consistent with Empirical Studies

Using transition state theory (Eyring equation) rates were extrapolatedfrom the calculated transition states (TS(1→2b) and TS(5→2a)), and foundthese were consistent with the myriad of published small molecule andprotein experimental data (see below), and the data presented here. Inparticular, the QM results provide mechanistic insight into howdecreasing the pKa of the leaving group sulfur (via sulfenic acid anioninstead of a sulfur anion) increased the overall reaction rate by twoseparate mechanisms (i.e., in addition to eliminating the ratedetermining thiol oxidation, cyclic thiosulfinates decrease the barrierof the new rate limiting step, thiolate-disulfide interchange).Specifically, a series of detailed (multi-concentration) kineticsexperiments was performed following the protocol of Singh et al. anddetermined the overall second order rate constant of cyclicthiosulfinate-mediated cross-linking product formation to be 1.5×104 M−1min−1 which, under our experimental conditions, extrapolates to apredicted half-life of 2.7 min for cross-linking SOD1 with1,2-dithiane-1-oxide.⁴⁹ Notably this value is consistent with half-lifefitted from FIG. 1 (2.2 min) and is consistent with the absolute rateextrapolated from the calculated value of 13.1 kcal/mol for thethiol-disulfide intermediate 2b. being the rate determining step. Thethiol-disulfide exchange reaction of oxidized DTT (cyclic disulfide) andreduced glutathione, which provides the closest analogy to the1,2-dithiane data presented here, has an extrapolated half-life of >200mins (rate constant of 1.8×102 M−1 min−1).⁵⁰ In fact, the rates observedand calculated for cyclic thiosulfinates were faster than any everreported for cyclic disulfides (to our knowledge, the fastest reportedrate of thiol-disulfide interchange is the reaction of Papain-S-SCH3with DTT which gives a rate constant of 3.3×103 M−1 min−1 and anextrapolated half-life of 12 min).⁵¹ These combined results demonstratethat thiosulfinates speed the overall rate both by eliminating the ratedetermining-S oxidation, and then by decreasing the new rate limitingstep, thiolate-disulfide interchange. It was noted that although thereported condensation reaction rates of thiolates and sulfenic acidsvary considerably, our experimentally determined rate constants are nearthe median of reported values, and are also consistent with the 11.0kcal/mol transition state (TS(3a,3b→4)) reported in as-reviewed FIG. 1.For example, Gupta et al. reported the rate constants of disulfideformation through sulfenic acid/thiolate condensation range from >106M−1 min−1 (Cys-SOH+Cys) to 1.3×103 M−1 min−1 (HSA-SOH+Cys), whichextrapolate to half-lives ranging from milliseconds to 30 mins,respectively, under our experimental conditions.⁵²

Thapa et al. highlighted the difficulty of computing pKa's of thiolswithout explicit solvation, and experimental rates of thiol-disulfideexchange generally decrease by three orders of magnitude in proticcompared to aprotic solvents, and by as much as nine orders of magnitudein water compared to the gas phase.⁵³⁻⁵⁴ Given these obstacles, and thatthe protein includes both protic and aprotic environments, continuummodel-based calculations for intermediates 3a and 3b were not reported.The experimental data required to estimate the pKa of this intermediatewas not obtained, which is therefore listed as the arbitrary value of 0kcal/mol.⁵⁵ Note, however, this value is consistent with theexperimentally determined rates, which imply that the energy ofintermediate 3a,3b cannot be higher than c.a. 3 kcal/mol, as well as thelack of observation of mono-thiol intermediates, which implies that itsenergy probably isn't lower than c.a. −3 kcal/mol.

Use of Cyclic Thiosulfinates for Nanoparticle, Dendrimer, and PolymerSyntheses

It was demonstrated that cyclic thiosulfinates could replace diene(e.g., bismaleimides) cross-linkers for nanoparticle, dendrimer, andpolymer syntheses and the results are shown in FIG. 14.

Briefly, polyethylene dithiol was mixed with either: no facilitator (−);trithiocyanuric acid; trimethylolpropane tris(3-mercaptopropionate); orpentaerythritol tetra(3-mercaptopropionate) and each mixture was treatedwith either 1,2-dithiane-1-oxo cross-linker or no cross-linker (−). Thered arrows in FIG. 14 indicate a reaction in which polymer formed andthe orange arrow indicate a reaction in which putative nanoparticlesformed. Poly(ethylene glycol) dithiol (PEG-DT, e.g., with average Mn of1,500) was mixed with 1,2-dithiane-1-oxo cross-linker to undergo a solidstate reaction to produce a transparent polymer.

It will be appreciated by those skilled in the art that the invention isnot restricted in its use to the particular application described.Neither is the present invention restricted in its preferred embodimentwith regard to the particular elements and/or features described ordepicted herein. It will be appreciated that the invention is notlimited to the embodiment or embodiments disclosed, but is capable ofnumerous rearrangements, modifications and substitutions withoutdeparting from the scope of the invention as set forth and defined bythe claims.

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1. A polymer derived from a first monomer and a first cross-linker,wherein the first monomer comprises at least two thiol functionalgroups; the first cross-linker comprises a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring.
 2. Thepolymer according to claim 1, wherein the first monomer is selected fromthe group consisting of a dithiol compound, a trithiol compound, atetrathiol compound, and a thiomer.
 3. The polymer according to claim 1,wherein the first monomer is selected from the group consisting of adithiol compound, a trithiol compound, a tetrathiol compound, ahexathiol compound, and an octathiol compound.
 4. The polymer accordingto any one of claims 1-3, wherein the first monomer is selected from thegroup consisting of dithiothreitol (DTT), 1,2-ethanedithiol,1,3-propanedithiol, 1,4-butanedithiol, 1,5-pentanedithiol,1,6-hexanedithiol, 1,7-heptanedithiol, 1,8-octanedithiol,1,9-nonanedithiol, 1,10-decanedithiol, 1,11-undecanedithiol,1,12-dodecanedithiol, 1,13-tridecanedithiol, 1,14-tetradecanedithiol,1,16-hexadecanedithiol, dithiolbutylamine (DTBA), tetra(ethylene glycol)dithiol, hexa(ethylene glycol) dithiol, 2-mercaptoethyl ether,2,2′-thiodiethanethiol, 2,2′-(ethylenedioxy)diethanethiol,propane-1,2,3-trithiol, trimethylolpropane tris(2-mercaptoacetate),trimethylolpropane tris(3-mercaptoacetate),3,3′,3″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(propane-1-thiol),4,4′,4″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(butane-1-thiol),pentaerythrityl tetrathiol, pentaerythritoltetrakis(3-mercaptopropionate), a peptide having a disulfide bond, and aprotein having a disulfide bond.
 5. The polymer according to any one ofclaims 1-3, wherein the first monomer is selected from the groupconsisting of poly(ethylene glycol) dithiol, 4arm-PEG2K-SH,4arm-PEG5K-SH, 4arm-PEG10K-SH, 4arm-PEG20K-SH, 4-arm poly(ethyleneoxide) thiol-terminated, 8arm-PEG10K-SH (hexaglyerol core),8arm-PEG10K-SH (tripentaerythritol core), 8arm-PEG20K-SH (hexaglyerolcore), 8arm-PEG20K-SH (tripentaerythritol core), and 8-arm poly(ethyleneoxide) thiol-terminated.
 6. The polymer according to claim 1, whereinthe first monomer is a peptide having a disulfide bond or a proteinhaving a disulfide bond.
 7. The polymer according to claim 6, whereinthe first monomer is a protein having a disulfide bond selected from thegroup consisting of a copper/zinc superoxide dismutase (SOD1) variant, aDJ-1 variant, an effector caspae, and iron-sulfur cluster assemblyenzyme (IscU).
 8. The polymer according to claim 7, wherein the firstmonomer is a familial Amyotrophic Lateral Sclerosis (fALS)-SOD1 variant.9. The polymer according to any one of claims 1-8, wherein W and Y areS.
 10. The polymer according to any one of claims 1-8, wherein W is Sand Y is S(O).
 11. The polymer according to any one of claims 1-8,wherein W and Y are Se.
 12. The polymer according to any one of claims1-8, wherein W is Se and Y is Se(O).
 13. The polymer according to anyone of claims 1-8, wherein W is Se and Y is S.
 14. The polymer accordingto any one of claims 1-8, wherein W is S and Y is Se.
 15. The polymeraccording to any one of claims 1-8, wherein W is Se and Y is S(O). 16.The polymer according to any one of claims 1-8, wherein W is S and Y isSe(O).
 17. The polymer according to any one of claims 1-16, wherein X is—(CR¹R²)_(n)—, wherein n is an integer selected from the groupconsisting of 1, 2, 3, 4, 5, 6, 7 and 8; and each R¹ and each R² on eachCR¹R² group is independently selected from the group consisting of H,OH, NO₂, CN, NH₂, optionally substituted C₁-C₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl,optionally substituted C₂-C₁₂heteroalkyl, optionally substitutedC₃-C₁₂cycloalkyl, optionally substituted C₂-C₁₂heterocycloalkyl,optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substitutedC₆-C₁₈ aryl, optionally substituted C₁-C₁₈heteroaryl, optionallysubstituted C₁-C₁₂alkyloxy, optionally substituted C₂-C₁₂alkenyloxy,optionally substituted C₂-C₁₂alkynyloxy, optionally substitutedC₂-C₁₂heteroalkyloxy, optionally substituted C₃-C₁₂cycloalkyloxy,optionally substituted C₃-C₁₂cycloalkenyloxy, optionally substitutedC₁-C₁₂heterocycloalkyloxy, optionally substitutedC₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-C₁₈aryloxy,optionally substituted C₁-C₁₈heteroaryloxy, optionally substitutedC₁-C₁₂alkylamino, CONR³R⁴, NR³COR⁴, NR³COOR⁴, NR³SO₂R⁴, NR³CONR³R⁴, andNR³R⁴; and wherein each R³ and R⁴ is independently selected from thegroup consisting of: H, optionally substituted C₁-C₁₂alkyl, optionallysubstituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl,optionally substituted C₂-C₁₂heteroalkyl, optionally substitutedC₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionallysubstituted C₂-C₁₂heterocycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, andoptionally substituted C₁-C₁₈heteroaryl.
 18. The polymer according toclaim 17, wherein n is an integer selected from the group consisting of3, 4, 5, and
 6. 19. The polymer according to claim 17 or 18, whereineach R¹ and each R² on each CR¹R² group is independently selected fromthe group consisting of H, OH, NO₂, CN, NH₂, optionally substitutedC₁-C₁₂alkyl, optionally substituted C₂-C₁₂heteroalkyl, optionallysubstituted C₃-C₁₂cycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkyl, optionally substituted C₁-C₁₂alkyloxy,optionally substituted C₂-C₁₂heteroalkyloxy, optionally substitutedC₃-C₁₂cycloalkyloxy, and optionally substitutedC₁-C₁₂heterocycloalkyloxy.
 20. The polymer according to any one ofclaims 17-19, wherein each R¹ and each R² on each CR¹R² group isindependently selected from the group consisting of H, OH, NO₂, CN, NH₂,optionally substituted C₁-C₁₂alkyl, optionally substitutedC₃-C₁₂cycloalkyl, optionally substituted C₁-C₁₂alkyloxy, and optionallysubstituted C₃-C₁₂cycloalkyloxy.
 21. The polymer according to any one ofclaims 1-8 and 17-20, wherein the first cross-linker comprises a moietyof Formula I selected from the group consisting of


22. A method for covalent coupling of molecules comprising providing aplurality of first molecules comprising a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring; andcontacting the first molecules with a plurality of second moleculescomprising at least one thiol functional group; thereby forming aplurality of covalent bonds between the S or Se atoms of the firstmolecule and the free thiol groups of the second molecule.
 23. A methodof coating a surface of an object, a device, or an assembly, comprisingthe steps of: (a) providing a surface of an object, a device, or anassembly; (b) contacting the surface with a plurality of first moleculescomprising a reactive moiety and a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring; therebyforming a plurality of covalent bonds between the surface and thereactive moieties of the first molecule to form a first monolayer. 24.The method according to claim 23 further comprising (c) contacting themonolayer with a second molecule comprising at least one thiolfunctional group, thereby forming a first bilayer, wherein a pluralityof covalent bonds are formed between the S or Se atoms of the firstmolecule and the free thiol groups of the second molecule.
 25. Themethod according to claim 24 further comprising (d) applying anelectrical potential, thereby reducing the covalent bonds, thus forminga second monolayer.
 26. The method according to claim 25 furthercomprising (f) providing an aqueous mixture comprising water and aplurality of metal ions, thereby forming a complex, comprising aplurality of metal-chelating groups of the second monolayer chelated tothe plurality of metal ions, thus forming a second bilayer.
 27. Themethod according to claim 26, wherein the plurality of metal-chelatinggroups are S atoms.
 28. The method according to claim 27, wherein theplurality of metal-chelating groups are Se atoms.
 29. The methodaccording to any one of claims 26-28, wherein the plurality of metalions are a plurality of metal cations.
 30. The method according to claim29, wherein the metal cation has a charge of +1.
 31. The methodaccording to claim 30, wherein the metal cation is a cation of Ag or Au.32. The method according to claim 29, wherein the metal cation has acharge of +2.
 33. The method according to claim 32, wherein the metalcation is a cation of Ca, Cd, Co, Cr, Cu, Er, Fe, Hg, Mg, Mn, Nb, Ni,Pb, Pd, Sc, Sn, Sr, V, or Zn.
 34. The method according to claim 29,wherein the metal cation has a charge of +3.
 35. The method according toclaim 34, wherein the metal cation is a cation of Au, Ce, Dy, Er, Eu,Fe, Gd, Ho, La, Lu, Nb, Nd, Pm, Pr, Sm, Tb, Tm, or Yb.
 36. The methodaccording to any one of claims 26-35 further comprising (f) contactingthe second bilayer with a plurality of third molecules comprising amoiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring; therebyforming a trilayer, wherein the plurality of third molecules arechelated to the plurality of metal ions.
 37. The method according to anyone of claims 22-36, wherein W and Y are S.
 38. The method according toany one of claims 22-36, wherein W is S and Y is S(O).
 39. The methodaccording to any one of claims 22-36, wherein W and Y are Se.
 40. Themethod according to any one of claims 22-36, wherein W is Se and Y isSe(O).
 41. The method according to any one of claims 22-36, wherein W isSe and Y is S.
 42. The method according to any one of claims 22-36,wherein W is S and Y is Se.
 43. The method according to any one ofclaims 22-36, wherein W is Se and Y is S(O).
 44. The method according toany one of claims 22-36, wherein W is S and Y is Se(O).
 45. The methodaccording to any one of claims 22-44, wherein X is —(CR¹R²)_(n)—,wherein n is an integer selected from the group consisting of 1, 2, 3,4, 5, 6, 7 and 8; and each R¹ and each R² on each CR¹R² group isindependently selected from the group consisting of H, OH, NO₂, CN, NH₂,optionally substituted C₁-C₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl, optionally substitutedC₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₂-C₁₂heterocycloalkyl, optionally substitutedC₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionallysubstituted C₁-C₁₈heteroaryl, optionally substituted C₁-C₁₂alkyloxy,optionally substituted C₂-C₁₂alkenyloxy, optionally substitutedC₂-C₁₂alkynyloxy, optionally substituted C₂-C₁₂heteroalkyloxy,optionally substituted C₃-C₁₂cycloalkyloxy, optionally substitutedC₃-C₁₂cycloalkenyloxy, optionally substituted C₁-C₁₂heterocycloalkyloxy,optionally substituted C₂-C₁₂heterocycloalkenyloxy, optionallysubstituted C₆-C₁₈aryloxy, optionally substituted C₁-C₁₈heteroaryloxy,optionally substituted C₁-C₁₂alkylamino, CONR³R⁴, NR³COR⁴, NR³COOR⁴,NR³SO₂R⁴, NR³CONR³R⁴, and NR³R⁴; and wherein each R³ and R⁴ isindependently selected from the group consisting of: H, optionallysubstituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl,optionally substituted C₂-C₁₂alkynyl, optionally substitutedC₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionallysubstituted C₃-C₁₂cycloalkenyl, optionally substitutedC₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl,optionally substituted C₆-C₁₈aryl, and optionally substitutedC₁-C₁₈heteroaryl.
 46. The method according to any one of claims 22-45,wherein the first molecule comprises a moiety of Formula I selected fromthe group consisting of


47. The method according to any one of claims 22 and 24-46, wherein thesecond molecule is selected from the group consisting of(11-mercaptoundecyl)-N,N,N-trimethylammonium bromide,(11-mercaptoundecyl)hexa(ethylene glycol),(11-mercaptoundecyl)tetra(ethylene glycol),1-(11-mercaptoundecyl)imidazole, 1-mercapto-2-propanol,11-(1H-pyrrol-1-yl)undecane-1-thiol, 11-(ferrocenyl)undecanethiol,11-amino-1-undecanethiol hydrochloride, 11-azido-1-undecanethiol,11-mercapto-1-undecanol, 11-mercaptoundecanamide, 11-mercaptoundecanoicacid, 11-mercaptoundecylhydroquinone, 11-mercaptoundecylphos-phonicacid, 12-mercaptododecanoic acid, 16-amino-1-hexadecanethiolhydrochloride, 16-mercaptohexadecanamide, 16-mercaptohexadecanoic acid,3-amino-1-propanethiol hydrochloride, 3-chloro-1-propanethiol,3-mercapto-1-propanol, 3-mercaptopropionic acid, 4-mercapto-1-butanol,6-(ferrocenyl)hexanethiol, 6-amino-1-hexanethiol hydrochloride,6-mercapto-1-hexanol, 6-mercaptohexanoic acid, 8-amino-1-octanethiolhydrochloride, 8-mercapto-1-octanol, 8-mercaptooctanoic acid,9-mercapto-1-nonanol, triethylene glycol mono-1-mercaptoundecyl ether,1-mercaptosuccinic acid, a peptide having a cysteine residue, a proteinhaving a cysteine residue, cysteamine, 1-thiohexitol, poly(ethyleneglycol) 2-mercaptoethyl ether acetic acid, poly(ethylene glycol) methylether thiol, 1-thioglycerol, 2-naphthalenethiol, biphenyl-4-thiol,3-amino-1,2,4-triazole-5-thiol, 5-(trifluoromethyl)pyridine-2-thiol,1-[2-(dimethylamino)ethyl]-1H-tetrazole-5-thiol, 1-propanethiol,1-butanethiol, 1-pentanethiol, 1-hexanethiol, 1-octanethiol,3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octanethiol, and γ-Glu-Cys.48. The method according to any one of claims 22 and 24-46, wherein thesecond molecule is selected from the group consisting of a dithiolcompound, a trithiol compound, a tetrathiol compound, and a thiomer. 49.The method according to any one of claims 22 and 24-46, wherein thesecond molecule is selected from the group consisting of a dithiolcompound, a trithiol compound, a tetrathiol compound, a hexathiolcompound, and an octathiol compound.
 50. The method according to any oneof claims 22, 24-46, 48, and 49, wherein the second molecule is selectedfrom the group consisting of DTT, 1,2-ethanedithiol, 1,3-propanedithiol,1,4-butanedithiol, 1,5-pentanedithiol, 1,6-hexanedithiol,1,7-heptanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,1,10-decanedithiol, 1,11-undecanedithiol, 1,12-dodecanedithiol,1,13-tridecanedithiol, 1,14-tetradecanedithiol, 1,16-hexadecanedithiol,DTBA, tetra(ethylene glycol) dithiol, hexa(ethylene glycol) dithiol,2-mercaptoethyl ether, 2,2′-thiodiethanethiol,2,2′-(ethylenedioxy)diethanethiol, propane-1,2,3-trithiol,trimethylolpropane tris(2-mercaptoacetate), trimethylolpropanetris(3-mercaptoacetate),3,3′,3″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(propane-1-thiol),4,4′,4″-((((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(propane-3,1-diyl))tris(sulfanediyl))tris(butane-1-thiol),pentaerythrityl tetrathiol, pentaerythritoltetrakis(3-mercaptopropionate), a peptide having a disulfide bond, and aprotein having a disulfide bond.
 51. The method according to any one ofclaims 22, 24-46, 48, and 49, wherein the second molecule is selectedfrom the group consisting of poly(ethylene glycol) dithiol,4arm-PEG2K-SH, 4arm-PEG5K-SH, 4arm-PEG10K-SH, 4arm-PEG20K-SH, 4-armpoly(ethylene oxide) thiol-terminated, 8arm-PEG10K-SH (hexaglyerolcore), 8arm-PEG10K-SH (tripentaerythritol core), 8arm-PEG20K-SH(hexaglyerol core), 8arm-PEG20K-SH (tripentaerythritol core), and 8-armpoly(ethylene oxide) thiol-terminated.
 52. The method according to anyone of claims 24-46, 48, and 49, wherein the second molecule is apeptide having a disulfide bond or a protein having a disulfide bond.53. The method according to claim 52, wherein the second molecule is aprotein having a disulfide bond selected from the group consisting of anSOD1 variant, a DJ-1 variant, an effector caspae, and IscU.
 54. Themethod according to claim 53, wherein the second molecule is a fALS-SOD1variant.
 55. A composition, wherein the composition comprises asubstrate and a coating material, wherein the coating material comprisesa first monolayer comprising a plurality of moieties of Formula I

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring; and thecoating material is covalently bonded to the substrate.
 56. Thecomposition according to claim 55, wherein the coating materialcomprises a first bilayer comprising a polymer derived from the moietiesof Formula I and a plurality of first monomers comprising at least onethiol functional group, wherein a plurality of covalent bonds are formedbetween the S or Se atoms of the plurality of moieties and the freethiol groups of the first monomer.
 57. The composition according toclaim 56, wherein the coating material comprises a second monolayercomprising a reduced form of the plurality of moieties of Formula I. 58.The composition according to claim 57, wherein the coating materialcomprises a second bilayer comprising a plurality of metal-chelatinggroups of the second monolayer chelated to a plurality of metal ions.59. The composition according to claim 58, wherein the plurality ofmetal-chelating groups are S atoms.
 60. The composition according toclaim 58, wherein the plurality of metal-chelating groups are Se atoms.61. The composition according to any one of claims 58-60, wherein theplurality of metal ions are a plurality of metal cations.
 62. Thecomposition according to claim 61, wherein the metal cation has a chargeof +1.
 63. The composition according to claim 62, wherein the metalcation is a cation of Ag or Au.
 64. The composition according to claim61, wherein the metal cation has a charge of +2.
 65. The compositionaccording to claim 64, wherein the metal cation is a cation of Ca, Cd,Co, Cr, Cu, Er, Fe, Hg, Mg, Mn, Nb, Ni, Pb, Pd, Sc, Sn, Sr, V, or Zn.66. The composition according to claim 61, wherein the metal cation hasa charge of +3.
 67. The composition according to claim 66, wherein themetal cation is a cation of Au, Ce, Dy, Er, Eu, Fe, Gd, Ho, La, Lu, Nb,Nd, Pm, Pr, Sm, Tb, Tm, or Yb.
 68. The composition according to any oneof claims 58-67, wherein the coating material comprises a trilayerfurther comprising a plurality of third molecules chelated to theplurality of metal ions, wherein the third molecules comprise a moietyof Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring.
 69. Thecomposition according to any one of claims 55-68, wherein the firstmonolayer comprises a plurality of moieties selected from the groupconsisting of


70. A method for transporting a molecule of interest across a cellmembrane comprising: (a) providing a functionalized molecule of interestcomprising a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring; and (b)contacting a cell of interest with the functionalized molecule ofinterest under conditions for the moiety of Formula I to reversibly bindto one or more cell membrane transport proteins, thereby facilitatingtransport of the molecule of interest across the cell membrane.
 71. Themethod according to claim 70, wherein the functionalized moleculecomprises a moiety of Formula I selected from the group consisting of


72. A method for treating a keratin-containing material, comprising: i)providing a plurality of first monomers, wherein the first monomer is akeratin-containing material sample comprising a plurality of disulfidebonds; ii) applying to the keratin-containing material sample for aperiod of time a mixture, comprising a reducing agent in a concentrationof about 0.1% by weight to about 15% by weight, thereby producing areduced keratin-containing material sample, wherein the reducedkeratin-containing material sample comprises a plurality of free thiolgroups; and iii) applying a first cross-linker to the reducedkeratin-containing material sample, wherein the first cross-linkercomprises a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with Wand Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring, therebyforming a plurality of covalent bonds between the free thiol groups andthe first cross-linkers.
 73. The method of claim 72, wherein thereducing agent is selected from the group consisting of ammoniumthioglycolate, L-cysteine, glutathione, ascorbic acid,beta-mercaptoethanol, 2-mercaptoethylamine, 2-mercaptoethylaminehydrochloride, dithiothreitol (DTT), thiolactic acid, thiosalicylicacid, tris-2-carboxyethylphospine hydrochloride (TCEP), sodiumhydrosulfite, sodium thiosulfate, potassium disulfite, sodium disulfite,sodium bisulfate, sodium bisulfite, ammonium bisulfite, thioglycolicacid, calcium thioglycolate, potassium thioglycolate, sodiumthioglycolate, cysteine hydrochloride, ammonium thiolactate,thioglycerin, mercaptoprpionic acid, glycerol thioglycolate anddithiolbutylamine (DTBA).
 74. A method for treating a keratin-containingmaterial, comprising: i) providing a plurality of first monomers,wherein the first monomer is a keratin-containing material samplecomprising a plurality of disulfide bonds; ii) applying to thekeratin-containing material sample for a period of time a mixture,comprising a first cross-linker, wherein the first cross-linkercomprises a moiety of Formula I:

wherein W is independently selected from the group consisting of S andSe; Y is independently selected from the group consisting of S, Se,S(O), Se(O), S(O)₂ and Se(O)₂; and X together with W and Y forms asubstituted or unsubstituted 3-10-membered heterocyclic ring, therebyforming a plurality of free thiol groups which react with the firstcross-linkers to form a plurality of covalent bonds between the freethiol groups and the first cross-linkers.
 75. The method of any one ofclaims 72-74, wherein the keratin-containing material is selected fromthe group consisting of hair, eyebrows, eyelashes, fingernails andtoenails.
 76. The method of any one of claims 72-74, wherein thekeratin-containing material is hair.